Borrelia diagnostics and screening methods

ABSTRACT

The present invention provides methods of detecting  Borrelia  species in a sample (e.g., a sample from a patient suspected of being infected). In particular, the present invention provides compositions and methods for detecting the presence of  Borrelia  proteins, nucleic acid sequences encoding these proteins, and subject antibodies to these proteins, where the proteins are selected from those listed in Table 3, including: BB0279 (FliL), BBK19, BBK07, BB0286 (FlbB), BBG33, BBL27, BBN34, BBP34, BBQ42, BBQ34, BBM34, BBN27, and BBH13.

The present application is a divisional of U.S. patent application Ser.No. 12/676,794, filed Jul. 15, 2010, now allowed, which is a §371 U.S.National Stage Entry of International Patent Application No.PCT/US2008/075613, filed Sep. 8, 2008, which claims priority to expiredU.S. Provisional Application Ser. No. 60/970,837, filed Sep. 7, 2007,each of which are herein incorporated by reference.

The invention was made with government support under grant numbersAI24424, AI065359, AI072872, LM007743, and AR20358 awarded by theNational Institutes of Health, and grant number MRI EIA-0321390 awardedby the National Science Foundation. The government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention relates to method of detecting Borrelia species ina sample (e.g., a sample from a patient suspected of being infected). Inparticular, the present invention provides compositions and methods fordetecting the presence of Borrelia proteins, nucleic acid sequencesencoding these proteins, and patient antibodies to these proteins, wherethe proteins are selected from those listed in Table 3, including:BB0279 (FliL), BBK19, BBK07, BB0286 (FlbB), BBG33, BBL27, BBN34, BBP34,BBQ42, BBQ34, BBM34, BBN27, and BBH13.

BACKGROUND OF THE INVENTION

Lyme disease is the most frequently reported arthropod-borne disease inthe United States and Europe (reviewed in ref (Steere, 2004).Serological assays are the most common laboratory tests used to confirmor support a diagnosis based on clinical features and epidemiologiccircumstances (reviewed in (Bunikis, 2002; Aguero-Rosenfeld, 2005).Direct detection of the organism by cultivation, histology of a biopsy,or by an approved and validated polymerase chain reaction assay isgenerally preferable to serological assays for definitive confirmationof a clinical diagnosis but these procedures are uncommon in practiceand not likely become widely used for the foreseeable future.

A clinical diagnosis of Lyme disease based on the observation of acharacteristic skin rash and suitable epidemiologic features (e.g.exposure to ticks in an endemic area during the season of transmission)can have high accuracy (Steere, 2004). But in the absence of a skin rash(˜20-30% of cases) diagnosis of early Lyme disease solely based onclinical and epidemiologic features is more difficult. Accuratediagnosis of early infection without the typical skin rash is important,because oral antibiotic treatment at this point is usually successfuland will prevent the more serious manifestations of disseminated diseaseand late disease. Serologic assays for late disseminated Lyme diseaseare also important to help confirm a clinical diagnosis ofpotentially-treatable chronic infection. But a commonly used, if notrecommended, practice is to use a serologic assay to “rule out” B.burgdorferi infection as an explanation of what may be long-standingsymptoms, such as chronic joint pain, headache, cognitive problems, andfatigue. For diagnosis of early infection, a sensitive test is desirableto identify the infection at the earliest and most easily treatablepoint of the infection. For diagnosis of late disease, high sensitivityis also desirable but improved specificity is especially importantbecause the test in practice is often applied in circumstances in whichthe a priori likelihood of B. burgdorferi infection is low (Bunikis,2002).

Currently available commercial assays in the United States are eitherbased on whole bacteria cell extracts, such as the enzyme-linkedimmunoabsorbent (ELISA) and Western blot assays, or on a single antigenELISA such as the C6 peptide of the VlsE protein (Aguero-Rosenfeld,2005). The whole cell assays are usually used as a 2-tiered test. First,a more sensitive, typically a whole cell ELISA, is used. This isfollowed by the more specific Western blot, if the ELISA is positive orequivocal (Control, 1997). Together these assays have served for yearsas the standard for serodiagnosis, but there remain trade-offs betweensensitivity and specificity to minimize false-positive results. Onedrawback of the 2-tiered, sequential test procedure is the time it takesand the greater expense for two assays. Another problem with whole cellassays is a lack of standardization between tests of differentmanufacturers. The variables include different strains of B. burgdorferithat are used, different conditions for cultivating the organisms, anddifferent methods for identifying the key antigens on blots.

Assays based on single proteins, such as the flagellin protein FlaB, orcombinations of recombinant proteins are available in Europe (Hansen,1988; Kaiser, 1999; Heikkila, 2003). In general, these have shownsensitivities and specificities approximately equivalent to the 2-tieredprocedure. The recombinant antigens used singly or in combination arethose that had been previously identified in whole cell Western blotassays using in-vitro cultivated cells. In the United States the mostcommon subunit assays use a single peptide (called C6) of the VlsEprotein or the full-length recombinant VlsE protein (Bacon, 2003). Insome test formulations these single antigen assays had sensitivity fordifferent stages of infection that was as good as the 2-tier procedureand better specificity (Lawrenz, 1999; Liang, 1999). But in other, morerecent studies, including some from Europe, either the specificity orsensitivity of single antigen assays was not as good as tests based ontwo or more antigens or a 2-tiered procedure (Peltomaa, 2004; Marangoni,2005; Goettner, 2005).

Perhaps the most important problem with currently available wholecell-based assays is that they utilize for their substrates bacteriathat have been grown in vitro. The accumulated evidence=unequivocallyshows that cells grown in vitro differ with respect to the expression ofseveral proteins from cells recovered from infected animals (Fikrig,1997; Gilmore, 2001; Salazar, 2005). While certain in vivo conditionscan be duplicated to some extent in vitro by altering growth conditions,such as pH or cell density, there remain many proteins that appear to beonly expressed in an infected animal or untreated patient.

SUMMARY OF THE INVENTION

The present invention provides methods of detecting Borrelia species ina sample (e.g., a sample from a patient suspected of being infected). Inparticular, the present invention provides compositions and methods fordetecting the presence of Borrelia proteins, nucleic acid sequencesencoding these proteins, and patient antibodies to these proteins, wherethe proteins are selected from those listed in Table 3, including:BB0279 (FliL), BBK19, BBK07, BB0286 (FlbB), BBG33, BBL27, BBN34, BBP34,BBQ42, BBQ34, BBM34, BBN27, and BBH13.

In some embodiments, the present invention provides methods of detectingBorrelia in a patient sample comprising: contacting a sample with anantibody or other agent configured to bind a molecule selected from thegroup consisting of: BB0279 (FliL), a BB0279 patient antibody, BBK19, aBBK19 patient antibody, BBK07, a BBK07 patient antibody, BB0286 (FlbB),a BB0286 patient antibody, BBG33, a BBG33 patient antibody, BBL27, aBBL27 patient antibody, BBN34, a BBN34 patient antibody, BBP34, a BBP34patient antibody, BBQ42, a BBQ42 patient antibody, BBQ34, a BBQ34patient antibody, BBM34, a BBM34 patient antibody, BBN27, a BBN27patient antibody, BBH13, and a BBH13 patient antibody.

In certain embodiments, the contacting is performed with the antibody ora fragment of the antibody. In further embodiments, the other agent isone of the molecules that is not an antibody (e.g., BB0279, BBK19,etc.), and the presence or absence of one or more of the patientantibodies is detected (e.g., a BB0279 patient antibody or BBK19 patientantibody).

In particular embodiments, the Borrelia bacteria detected is Borreliaburgdorferi. In other embodiments, the Borrelia is Borrelia afzelii orBorrelia garinii. In certain embodiments, the Borrelia bacteria detectedis selected from: Borrelia afzelii; Borrelia anserina; Borreliaburgdorferi; Borrelia garinii; Borrelia hermsii; Borrelia recurrentis;and Borrelia valaisiana.

In some embodiments, the present invention provides methods of detectingBorrelia in a sample comprising: contacting a sample with an nucleicacid sequence or nucleic acid sequences configured to detect a targetnucleic acid sequence selected from the group consisting of: BB0279(FliL), BBK19, BBK07, BB0286 (FlbB), BBG33, BBL27, BBN34, BBP34, BBQ42,BBQ34, BBM34, BBN27, and BBH13.

In certain embodiments, the nucleic acid sequence is a probe thatcomprises a nucleotide sequence selected from the group consisting of:SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:115,SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:125, SEQ IDNO:126, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:143, SEQ ID NO:144, SEQID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:161,SEQ ID NO:162, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:185, SEQ IDNO:186, SEQ ID NO:191, SEQ ID NO:192 or any of the nucleic acidsequences (or portions thereof) shown in the accession numbers in Table3.

In other embodiments, the nucleic acid sequences are a primer pairselected from the group consisting of: SEQ ID NO:15 and SEQ ID NO:16;SEQ ID NO:19 and SEQ ID NO:20; SEQ ID NO:115 and SEQ ID NO:116; SEQ IDNO:119 and SEQ ID NO:120; SEQ ID NO:125 and SEQ ID NO:126; SEQ ID NO:131and SEQ ID NO:132; SEQ ID NO:143 and SEQ ID NO:144; SEQ ID NO:151 andSEQ ID NO:152; SEQ ID NO:157 and SEQ ID NO:158; SEQ ID NO:161 and SEQ IDNO:162; SEQ ID NO:173 and SEQ ID NO:174; SEQ ID NO:185 and SEQ IDNO:186; and SEQ ID NO:191 and SEQ ID NO:192.

In particular embodiments, the present invention provides methods ofvaccinating a person against Borrelia infection, comprising:administering a composition to a patient comprising an isolated proteinselected from the group consisting of: BB0279 (FliL), BBK19, BBK07,BB0286 (FlbB), BBG33, BBL27, BBN34, BBP34, BBQ42, BBQ34, BBM34, BBN27,and BBH13.

In certain embodiments, the present invention provides compositionssuitable for injection to a human (or domesticated animal) comprising:i) an adjuvant and/or physiological tolerable buffer, and ii) anisolated protein selected from the group consisting of: BB0279 (FliL),BBK19, BBK07, BB0286 (FlbB), BBG33, BBL27, BBN34, BBP34, BBQ42, BBQ34,BBM34, BBN27, and BBH13.

In other embodiments, the present invention provides methods ofdetecting Borrelia in a sample comprising: contacting a sample with anantibody or other agent configured to bind a molecule selected from anantigen recited in Table 3 or an antibody to an antigen in Table 3.

In some embodiments, the present invention provides methods of detectingBorrelia in a sample comprising: contacting a sample with an antibody orother agent configured to bind a molecule selected from the groupconsisting of: BBG33 or antibody thereof; BB0279 or antibody thereof;BBL27 or antibody thereof; BBN34 or antibody thereof; BBP34 or antibodythereof; BBQ42 or antibody thereof; BBQ34 or antibody thereof; BBM34 orantibody thereof; BBN27 or antibody thereof; BBH13 or antibody thereof;BB034 or antibody thereof; BBQ03 or antibody thereof; BBN11 or antibodythereof; OspC_A or antibody thereof; BBO39 or antibody thereof; BBF03 orantibody thereof; BBK19 or antibody thereof; BBI42 or antibody thereof;BBB14 or antibody thereof; BB0348 or antibody thereof; BBH06 or antibodythereof; BBN38 or antibody thereof; BB0215 or antibody thereof; OspC_Kor antibody thereof; BBA36 or antibody thereof; BBL40 or antibodythereof; BB0359 or antibody thereof; BBR42 or antibody thereof; BBJ24 orantibody thereof; BB0543 or antibody thereof; BB0774 or antibodythereof; BB0844 or antibody thereof; BBN39 or antibody thereof; BBK12 orantibody thereof; BBA07 or antibody thereof; BBK07 or antibody thereof;BBA57 or antibody thereof; BB0323 or antibody thereof; BB0681 orantibody thereof; BBA03 or antibody thereof; BBB09 or antibody thereof;BB0238 or antibody thereof; BBA48 or antibody thereof; BB0408 orantibody thereof; BBK53 or antibody thereof; BBR35 or antibody thereof;BBS41 or antibody thereof; BB0286 or antibody thereof; BB0385 orantibody thereof; and BBG18 or antibody thereof.

In particular embodiments, the present invention provides methods ofdetecting Borrelia in a sample comprising: contacting a sample with anucleic acid sequence or nucleic acid sequences configured to detect atleast one target nucleic acid sequence of an antigen recited in Table 3.In some embodiments, the at least one target nucleic acid sequence isselected from the group consisting of: BBG33; BB0279; BBL27; BBN34;BBP34; BBQ42; BBQ34; BBM34; BBN27; BBH13; BBO34; BBQ03; BBN11; OspC_A;BBO39; BBF03; BBK19; BBI42; BBB14; BB0348; BBH06; BBN38; BB0215; OspC_K;BBA36; BBL40; BB0359; BBR42; BBJ24; BB0543; BB0774; BB0844; BBN39;BBK12; BBA07; BBK07; BBA57; BB0323; BB0681; BBA03; BBB09; BB0238; BBA48;BB0408; BBK53; BBR35; BBS41; BB0286; BB0385; and BBG18. In furtherembodiments, the nucleic acid sequences comprises at least one nucleicacid sequence selected from SEQ ID NOs:1-202.

In some embodiments, the present invention provides methods ofvaccinating a person against Borrelia, comprising: administering acomposition to a patient comprising at least one isolated protein fromTable 3. In particular embodiments, the at least one isolated protein isselected from the group consisting of: BBG33; BB0279; BBL27; BBN34;BBP34; BBQ42; BBQ34; BBM34; BBN27; BBH13; BB034; BBQ03; BBN11; OspC_A;BBO39; BBF03; BBK19; BBI42; BBB14; BB0348; BBH06; BBN38; BB0215; OspC_K;BBA36; BBL40; BB0359; BBR42; BBJ24; BB0543; BB0774; BB0844; BBN39;BBK12; BBA07; BBK07; BBA57; BB0323; BB0681; BBA03; BBB09; BB0238; BBA48;BB0408; BBK53; BBR35; BBS41; BB0286; BB0385; and BBG18.

In additional embodiments, the present invention provides compositionssuitable for injection to a human, or domesticated animal, comprising:i) an adjuvant and/or physiological tolerable buffer, and ii) anisolated protein from Table 3. In particular embodiments, the at leastone isolated protein is selected from the group consisting of: BBG33;BB0279; BBL27; BBN34; BBP34; BBQ42; BBQ34; BBM34; BBN27; BBH13; BBO34;BBQ03; BBN11; OspC_A; BBO39; BBF03; BBK19; BBI42; BBB14; BB0348; BBH06;BBN38; BB0215; OspC_K; BBA36; BBL40; BB0359; BBR42; BBJ24; BB0543;BB0774; BB0844; BBN39; BBK12; BBA07; BBK07; BBA57; BB0323; BB0681;BBA03; BBB09; BB0238; BBA48; BB0408; BBK53; BBR35; BBS41; BB0286;BB0385; and BBG18.

In some embodiments, the present invention provides methods of detectingBorrelia in a sample comprising: contacting a sample with an antibody orother agent configured to bind a molecule selected from the groupconsisting of: BBK07, a BBK07 ortholog, a BBK07 antibody, BBK12, a BBK12ortholog, a BBK12 antibody, BBK19, a BBK19 ortholog, a BBK antibody,FliL, a FliL ortholog, a FliL antibody, FlbB, a FlbB ortholog, or a FlbBantibody.

In other embodiments, the present invention provides methods ofdetecting Borrelia in a sample comprising: contacting a sample with annucleic acid sequence or nucleic acid sequences configured to detect atarget nucleic acid sequence selected from the group consisting of:bbk07, a bbk07 ortholog, bbk12, a bbk12 ortholog, bbk19, a bbk19ortholog, flil, a flil ortholog, flbb, or a flbbB ortholog.

In certain embodiments, the present invention provides methods forvaccinating a subject (e.g., a person) against Borrelia, comprising:administering a composition to a patient comprising an isolated proteinselected from the group consisting of: BBK07, a BBK07 ortholog, BBK12, aBBK12 ortholog, BBK19, a BBK19 ortholog, FliL, a FliL ortholog, FlbB, ora FlbB ortholog.

In further embodiments, the present invention provides compositionssuitable for injection to an animal (e.g., human) comprising: i) anadjuvant and/or physiological tolerable buffer, and ii) an isolatedprotein selected from the group consisting of: BBK07, a BBK07 ortholog,BBK12, a BBK12 ortholog, BBK19, a BBK19 ortholog, FliL, a FliL ortholog,FlbB, or a FlbB ortholog.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overview of genome-wide proteome array results obtainedwith sera from humans with LB as described in Example 1. The graphs aretwo sets of frequency histograms and scatter plots of binding ofantibodies in FIG. 1A and (upper panel) and FIG. 1B (lower panel) serafrom controls and patients with early or later LB to an array ofrecombinant proteins produced in vitro from a total of 1,293 B.burgdorferi ORFs (1,292 ORFs from strain B31 and 1 ORF from strain 297).In the frequency histograms the x axes indicate relative log₁₀ intensityvalues, and the y axes indicate the relative counts on an intervalscale. In the scatter plots, both the x and y axes indicate relativelog₁₀ intensity values. The distributions in the frequency histogramsare indicated along with the medians of log₁₀ intensity values with 95%confidence intervals. The scatter plots include two-sided P valuesobtained by using an exact Wilcoxon signed rank test.

FIG. 2 shows a two-color display of a Euclidian distance clusteranalysis of a small array with immunogenic Orfs of B. burgdorferi asdescribed in Example 1. The array was incubated with 12 sera fromindividuals with later LB (panel 1) or three control sera. In additionto selected Orfs from Table 1, the following three proteins were used:BB0383 (BmpA), BB0744 (P83/100), and BBA24 (DbpA). The Orf designationsdo not include “BB.” Protein names and hypothetical proteins (HP), aswell as the PFs, are indicated on the right. Clusters are indicated onthe left. The levels of bootstrap support (1,000 iterations) are asfollows: orange (dark gray), >50%; yellow (light gray), >60%; and black,100%. A scale for log₁₀ intensity values is at the bottom.

FIG. 3 shows scatter plots of array intensity values normalized in unitsof SDs above or below the mean for the controls of each panel. Each plotshows values for pairs of selected Orfs reacted with sera of controls(dark gray circles) and patients with early LB (blue multiplicationsigns) or later LB (light gray X's) of panels 1 and 2. The coefficientsof determination (R²) for all plots, as well as the linear regressionequations for the upper two plots, are shown. The levels of identity ofaligned amino acid sequences of the three pairs of homologous proteinsare indicated; BBK07 and BBA25 are not significantly (NS) similar.

FIG. 4 shows a Western blot analysis of purified recombinant proteinsencoded by ORFs BBA25 (DbpB), BBK12, BB279 (FliL), and BB283 (FlgE)incubated with sera of 17 patients with later LB or five panel controlsas described in Example 1. Binding of antibody was detected withalkaline phosphatase-labeled secondary antibody to human IgG asdescribed in the text.

FIG. 5 shows binding of antibodies in human LB sera to purified proteinson arrays as described in Example 1. The plots are box-whisker plots oflog-transformed intensity values for the binding of panel 1 sera frompatients with later LB (n=17) or controls (n=5) with purifiedrecombinant proteins encoded by ORFs BBA25, BBG33, BBK12, and BB283 atconcentrations of 0.03 mg/ml (dark gray), 0.1 mg/ml (medium gray), 0.3(light gray), and 0.9 mg/ml (black). Each box indicates the first andthird quartiles, and the line inside the box is the median. The 1.5×interquartile range is indicated by the vertical line bisecting the box,and values outside this range are indicated by asterisks and by circles.

FIG. 6 shows a Western blot analysis, from Example 1, of whole-celllysates of low-passage and high-passage B. burgdorferi strain B31 withmouse antiserum to recombinant BBK12 or with murine monoclonalantibodies to BBA15 (OspA) or BB0147 (FlaB).

FIG. 7 shows an exemplary estimation of the number of immunogens forassays with a particular level of high sensitivity and specificity. Thegraphs show four receiver operating characteristic curves for nonlinearclassifiers with different sets of Orfs and the effect of increasing theamounts of uniform Gaussian noise with a mean of 0 and an SD of 5, 10,25, 75, or 150. The antigens in sets containing 2, 5, 25, and 45antigens were selected in order of their ranking by theBayes-regularized analysis. The solid lines indicate the average withstandard error over cross-validation runs calculated at stepped(1—specificity) points. The error bars indicate 95% confidenceintervals. The dotted lines indicate the performance for each of 10threefold cross-validation iterations.

FIG. 8 shows a comparison of observed results with expected results fromsimulations: counts of Orfs in arrays with LB sera that were 3 SDs abovethe mean of control sera one or more times. Results for 39 panel 1 and 2sera from patients with later LB are compared with mean counts (with 95%confidence intervals) for four simulation runs with random linkages. Thenumbers of Orfs that exceeded the 3-SD cutoff one to seven times areindicated next to the corresponding symbols for the random linkagesimulation.

DEFINITIONS

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular antibody. When a protein orfragment of a protein (e.g., those described by accession number inTable 3) is used to immunize a host animal, numerous regions of theprotein may induce the production of antibodies which bind specificallyto a given region or three-dimensional structure on the protein; theseregions or structures are referred to as “antigenic determinants”. Anantigenic determinant may compete with the intact antigen (i.e., the“immunogen” used to elicit the immune response) for binding to anantibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “subject suspected of being infected with aBorrelia species” refers to a subject that presents one or more symptomsindicative of such infection (see, e.g., NIH guidelines for suchinfections). A subject suspected of being infected with Borrelia species(e.g., burgdorferi) may also have one or more risk factors (e.g.,exposure to ticks). A subject suspected of infection generally not beentested for such infection.

A “patient antibody,” as used herein, is an antibody generated in apatient (e.g., human) as a result of infection with a Borrelia bacteria.In other words, it is the patient's own antibodies generated as a resultof infection. Such antibodies provide evidence of infection and aretherefore useful to detect in order to provide a diagnosis of Borreliainfection.

As used herein, the term “instructions for using said kit for detectingBorrelia infection in said subject” includes instructions for using thereagents contained in the kit for the detection and characterization ofBorrelia infection in a sample from a subject. In some embodiments, theinstructions further comprise the statement of intended use required bythe U.S. Food and Drug Administration (FDA) in labeling in vitrodiagnostic products. The present invention contemplates kits withreagents for detecting Borrelia infection, including antibodies to theantigens recited in Table 3, and nucleic acids sequences (e.g., primerpairs from Table 4).

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.Exemplary primers for detecting the Borrelia target nucleic acids of thepresent invention are provided in Table 4, which contains 101 primerpairs (SEQ ID NOs:1-202). One of skill in the art could design similarprimers given that the nucleic acid sequences are known in the art forthe Borrelia antigens (Table 3 useful nucleic acid sequence accessionnumbers).

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to at least a portion ofanother oligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label. The primers listed in Table 4could also be used as probes (e.g., by labeling these sequences) todetect Borrelia antigens.

As used herein the term “portion” when in reference to a nucleotidesequence (as in “a portion of a given nucleotide sequence”) refers tofragments of that sequence. The fragments may range in size from fournucleotides to the entire nucleotide sequence minus one nucleotide (10nucleotides, 20, 30, 40, 50, 100, 200, etc.).

DESCRIPTION OF THE INVENTION

The present invention provides methods of detecting Borrelia species ina sample (e.g., a sample from a patient suspected of being infected). Inparticular, the present invention provides compositions and methods fordetecting the presence of Borrelia proteins, nucleic acid sequencesencoding these proteins, and patient antibodies to these proteins, wherethe proteins are selected from those listed in Table 3, including:BB0279 (FliL), BBK19, BBK07, BB0286 (FlbB), BBG33, BBL27, BBN34, BBP34,BBQ42, BBQ34, BBM34, BBN27, and BBH13.

I. Borrelia Antigens

The present invention provides numerous proteins and nucleic acidtargets that can be detected in order to diagnose Borrelia infection.Table 3 below lists the ORFs that were found to be antigenic during thedevelopment of the present invention. Table 3 also lists the accessionnumbers where the protein and nucleic acid sequences for these antigenscan be found. These accession numbers allow one skilled in the art toeasily design probes and primers to the corresponding nucleic acidsequences. These accession numbers (herein incorporated by reference asif fully set forth herein) also allow one of skill in the art to expressthese proteins in order to generate antibodies and antibody fragmentsuseful for detecting Borrelia infection.

TABLE 3 Accession Nos. for Nucleic and Amino ORF SEO ID NO. Deduced geneproduct Acid Sequences BB0056 212 Phosphoglycerate kinase NC_001318BB0108 213 Peptidylprolyl isomerase NC_001318 BB0181 214 Flagellarhook-associated protein (FlgK) NC_001318 BB0215 215 Phosphate ABCtransporter (PstS) NC_001318 BB0238 216 Hypothetical protein NC_001318BB0260 217 Hypothetical protein NC_001318 BB0279 218 Flagellar protein(FliL) NC_001318 BB0286 219 Flagellar protein (FlbB) NC_001318 BB0323220 Hypothetical protein NC_001318 BB0337 221 Enolase NC_001318 BB0348222 Pyruvate kinase NC_001318 BB0359 223 Carboxy-terminal proteaseNC_001318 BB0385 224 Basic membrane protein D (BmpD) NC_001318 BB0408225 Phosphotransferase system, fructose-specific IIABC NC_001318 BB0476226 Translation elongation factor TU (Tut) NC_001318 BB0543 227Hypothetical protein NC_001318 BB0652 228 Protein export protein (SecD)NC_001318 BB0668 229 Flagellar filament outer layer protein (FlaA)NC_001318 BB0681 230 Methyl-accepting chemotaxis protein NC_001318BB0751 231 Hypothetical protein NC_001318 BB0772 232 Flagellar P-ringprotein (FlgI) NC_001318 BB0774 233 Flagellar basal body cord protein(FlgG) NC_001318 BB0805 234 Polyribonucleotidyltransferase (PnpA)NC_001318 BB0811 235 Hypothetical protein (COG1413) NC_001318 BB0844 236Hypothetical protein NC_001318 BBA03 237 Hypothetical protein NC_001857BBA07 238 Hypothetical protein NC_001857 BBA19 239 Hypothetical proteinNC_001857 BBA36 240 Hypothetical protein NC_001857 BBA40 241Hypothetical protein NC_001857 BBA48 242 Hypothetical protein NC_001857BBA57 243 Hypothetical protein NC_001857 BBB09 244 Hypothetical proteinNC_001903 BBB14 245 Hypothetical protein NC_001903 BBC03 246Hypothetical protein NC_001904 BBE09 247 Hypothetical protein NC_001850BBF03 248 BdrS (BdrF1) NC_001851 BBG18 249 Hypothetical proteinNC_001852 BBG33 250 BdrT (BdrF2) NC_001852 BBH06 251 Hypotheticalprotein NC_001853 BBH13 252 BdrU (BdrF3) NC_001853 BB142 253Hypothetical protein NC_001854 BBJ24 254 Hypothetical protein NC_001856BBK07 255 Hypothetical protein NC_001855 BBK12 256 Hypothetical proteinNC_001855 BBK13 257 Hypothetical protein (COG2859) NC_001855 BBK19 258Hypothetical protein NC_001855 BBK23 259 Hypothetical protein NC_001855BBK52 260 “P23” NC_001855 BBK53 261 Hypothetical protein NC_001855 BBL03262 Hypothetical protein NC_000953 BBL27 263 BdrO (BdrE1) NC_000953BBL39 264 ErpN (CRASP-5) NC_000953 BBL40 265 ErpO NC_000953 BBM34 266BdrK (BdrD2) NC_000951 BBM36 267 Hypothetical protein NC_000951 BBN11268 Hypothetical protein NC_000954 BBN27 269 BdrR (BdrE2) NC_000954BBN34 270 BdrQ (BdrD10) NC_000954 BBN38 271 ErpP (CRASP-3) NC_000954BBN39 272 ErpQ NC_000954 BBO34 273 BdrM (BdrD3) NC_000952 BBO39 274 ErpLNC_000952 BBO40 275 ErpM NC_000952 BBP34 276 BdrA (BdrD4) NC_000948BBP39 277 ErpB NC_000948 BBQ03 278 Hypothetical protein AE001584 BBQ04279 Hypothetical protein AE001584 BBQ13 280 Hypothetical proteinAE001584 BBQ19 281 Hypothetical protein AE001584 BBQ34 282 BdrW (BdrE6)AE001584 BBQ40 283 Partition protein AE001584 BBQ42 284 BdrV (BdrD5)AE001584 BBR12 285 Hypothetical protein NC_000950 BBR35 286 BdrGNC_000950 BBR42 287 ErpY NC_000950 BBS41 288 ErpG NC_000949

II. Detection of Borrelia Infection

In some embodiments, the present invention provides methods fordetection of the Borrelia antigens listed in Table 3. In someembodiments, expression is detected in bodily fluids (e.g., includingbut not limited to, plasma, serum, whole blood, mucus, and urine). Incertain embodiments, multiple antigens are detected (e.g., two or moreantigens from Table 3 or one antigen from Table 3 and one antigenpresently known in the art). In certain embodiments, at least 2 . . . 5. . . 10 . . . 20 . . . 35 . . . 50 . . . or 100 antigens are detectedfrom a single patient sample.

In some embodiments, the presence of a Table 3 Borrelia antigen is usedto provide a prognosis to a subject. The information provided is alsoused to direct the course of treatment.

1. Detection of Nucleic Acid

In some embodiments, detection of Table 3 Borrelia antigens are detectedby measuring the existence of nucleic acid encoding such antigens in apatient sample. Table 3 lists the accession numbers for each of theantigens which allows one of skill in the art to design primers andprobes to such sequences. Exemplary primers for each of these antigensare shown in Table 4.

In some embodiments, nucleic acid is detected by Northern blot analysis.Northern blot analysis involves the separation of nucleic acid andhybridization of a complementary labeled probe.

In still further embodiments, nuclei acid is detected by hybridizationto an oligonucleotide probe). A variety of hybridization assays using avariety of technologies for hybridization and detection are available.For example, in some embodiments, TaqMan assay (PE Biosystems, FosterCity, Calif.; See e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each ofwhich is herein incorporated by reference) is utilized. The assay isperformed during a PCR reaction. The TaqMan assay exploits the 5′-3′exonuclease activity of the AMPLITAQ GOLD DNA polymerase. A probeconsisting of an oligonucleotide with a 5′-reporter dye (e.g., afluorescent dye) and a 3′-quencher dye is included in the PCR reaction.During PCR, if the probe is bound to its target, the 5′-3′ nucleolyticactivity of the AMPLITAQ GOLD polymerase cleaves the probe between thereporter and the quencher dye. The separation of the reporter dye fromthe quencher dye results in an increase of fluorescence. The signalaccumulates with each cycle of PCR and can be monitored with afluorimeter.

In other embodiments, nucleic acid is detected using a detection assayincluding, but not limited to, enzyme mismatch cleavage methods (e.g.,Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, hereinincorporated by reference in their entireties); polymerase chainreaction; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos.5,849,481, 5,710,264, 5,124,246, and 5,624,802, herein incorporated byreference in their entireties); rolling circle replication (e.g., U.S.Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, herein incorporated byreference in their entireties); NASBA (e.g., U.S. Pat. No. 5,409,818,herein incorporated by reference in its entirety); molecular beacontechnology (e.g., U.S. Pat. No. 6,150,097, herein incorporated byreference in its entirety); E-sensor technology (Motorola, U.S. Pat.Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, herein incorporatedby reference in their entireties); cycling probe technology (e.g., U.S.Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, herein incorporated byreference in their entireties); Dade Behring signal amplificationmethods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230,5,882,867, and 5,792,614, herein incorporated by reference in theirentireties); ligase chain reaction (Barnay Proc. Natl. Acad. Sci USA 88,189-93 (1991)); FULL-VELOCITY assays; and sandwich hybridization methods(e.g., U.S. Pat. No. 5,288,609, herein incorporated by reference in itsentirety). In other embodiments, the detection assay employed is theINVADER assay (Third Wave Technologies) which is described in U.S. Pat.Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, and 6,090,543, WO97/27214 WO 98/42873, Lyamichev et al., Nat. Biotech., 17:292 (1999),Hall et al., PNAS, USA, 97:8272 (2000), each of which is hereinincorporated by reference in their entirety for all purposes).

2. Detection of Protein

In some embodiments, the proteins expressed by the ORFs listed in Table3 are detected. Protein expression can be detected by any suitablemethod. In some embodiments, proteins are detected byimmunohistochemistry. In other embodiments, proteins are detected bytheir binding to an antibody raised against the protein. The generationof antibodies is described below.

Antibody binding is detected by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In certain embodiments, antibody binding is detected by detecting alabel on the primary antibody. In another embodiment, the primaryantibody is detected by detecting binding of a secondary antibody orreagent to the primary antibody. In a further embodiment, the secondaryantibody is labeled. Many methods are known in the art for detectingbinding in an immunoassay and are within the scope of the presentinvention.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. In other embodiments, theimmunoassay described in U.S. Pat. Nos. 5,599,677 and 5,672,480 (each ofwhich is herein incorporated by reference) is utilized.

3. Antibodies and Antibody Fragments

The present invention provides isolated antibodies and antibodyfragments against the Borrelia proteins recited in Table 3. Suchantibodies and antibody fragments can be used, for example, indiagnostic and therapeutic methods. The antibody, or antibody fragment,can be any monoclonal or polyclonal antibody that specifically recognizeBorrelia antigens recited in Table 3. In some embodiments, the presentinvention provides monoclonal antibodies, or fragments thereof, thatspecifically bind to Borrelia antigens recited in Table 3. In someembodiments, the monoclonal antibodies, or fragments thereof, arechimeric or humanized antibodies. In other embodiments, the monoclonalantibodies, or fragments thereof, are human antibodies.

The antibodies of the present invention find use in experimental,diagnostic and therapeutic methods. In certain embodiments, theantibodies of the present invention are used to detect the presence orabsence of Borrelia proteins in a sample from a patient.

Polyclonal antibodies can be prepared by any known method. Polyclonalantibodies can be raised by immunizing an animal (e.g. a rabbit, rat,mouse, donkey, etc) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (a purified peptide fragment,full-length recombinant protein, fusion protein, etc., from Table 3)optionally conjugated to keyhole limpet hemocyanin (KLH), serum albumin,etc. diluted in sterile saline and combined with an adjuvant (e.g.Complete or Incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized. Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Alternatively, lymphocytes can be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells. Hybridomas that produce monoclonal antibodiesdirected specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assaysuch as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA) can then be propagated either in vitro culture using standardmethods (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986) or in vivo as ascites tumors in an animal. Themonoclonal antibodies can then be purified from the culture medium orascites fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated, such as from mature B-cellsor hybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries as described (McCafferty et al., 1990, Nature,348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks etal., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In one embodiment, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In otherembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Furthermore,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of a monoclonal antibody.

In some embodiments, of the present invention the monoclonal antibodyagainst a Borrelia antigen from Table 3 is a humanized antibody.Humanized antibodies are antibodies that contain minimal sequences fromnon-human (e.g., murine) antibodies within the variable regions. Suchantibodies are used therapeutically to reduce antigenicity and HAMA(human anti-mouse antibody) responses when administered to a humansubject. In practice, humanized antibodies are typically humanantibodies with minimum to no non-human sequences. A human antibody isan antibody produced by a human or an antibody having an amino acidsequence corresponding to an antibody produced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDR of a humanantibody with that of a non-human antibody (e.g. mouse, rat, rabbit,hamster, etc.) having the desired specificity, affinity, and capability(Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988,Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536).The humanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability.

Human antibodies can be directly prepared using various techniques knownin the art Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, for example, Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat.No. 5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies (Vaughan etal., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS,95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Markset al., 1991, J. Mol. Biol., 222:581). Humanized antibodies can also bemade in transgenic mice containing human immunoglobulin loci that arecapable upon immunization of producing the full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Thisapproach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and 5,661,016.

This invention also encompasses bispecific antibodies. Bispecificantibodies are antibodies that are capable of specifically recognizingand binding at least two different epitopes.

Bispecific antibodies can be intact antibodies or antibody fragments.Techniques for making bispecific antibodies are common in the art(Millstein et al., 1983, Nature 305:537-539; Brennan et al., 1985,Science 229:81; Suresh et al, 1986, Methods in Enzymol. 121:120;Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalaby et al., 1992, J.Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol. 148:1547-1553;Gruber et al., 1994, J. Immunol. 152:5368; and U.S. Pat. No. 5,731,168).

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. Various techniques are known for theproduction of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117 and Brennan et al., 1985, Science, 229:81). However, thesefragments are now typically produced directly by recombinant host cellsas described above. Thus Fab, Fv, and scFv antibody fragments can all beexpressed in and secreted from E. coli or other host cells, thusallowing the production of large amounts of these fragments.Alternatively, such antibody fragments can be isolated from the antibodyphage libraries discussed above. The antibody fragment can also belinear antibodies as described in U.S. Pat. No. 5,641,870, for example,and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

It may further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

III. Treatment for Infection

In certain embodiments, after a patient has been diagnosed with Borreliainfection, that patient is administered appropriate antibiotics.However, certain patients may be referactory to antibiotic treatment. Insuch situations, other treatments are employed, such as using antibodiesto one or more of the antigens described in Table 3.

In some embodiments, the present invention provides antibodies thatproteins from Table 3. Any suitable antibody (e.g., monoclonal,polyclonal, or synthetic) can be utilized in the therapeutic methodsdisclosed herein. In some embodiments, the antibodies used for therapyare humanized antibodies. Methods for humanizing antibodies are wellknown in the art (See e.g., U.S. Pat. Nos. 6,180,370, 5,585,089,6,054,297, and 5,565,332; each of which is herein incorporated byreference).

In some embodiments, the antibody is conjugated to a cytotoxic agent.For certain applications, it is envisioned that the therapeutic agentswill be pharmacologic agents that will serve as useful agents forattachment to antibodies, particularly cytotoxic or otherwiseanticellular agents having the ability to kill Borrelia bacteria. Thepresent invention contemplates the use of any pharmacologic agent thatcan be conjugated to an antibody, and delivered in active form.

IV. BBK07, BBK12, and BBK19

The present invention provides compositions comprising proteinsequences, as well as the DNA sequences encoding them, of threeproteins, BBK07, BBKI2, and BBK19 (“the proteins”), of the Lyme diseaseagent Borrelia burgdorferi and deduced proteins of other pathogenicBorrelia species that are orthologous to these three proteins. Incertain embodiments, the present invention provides diagnostic tests forantibodies to Borrelia burgdorferi or other pathogenic Borrelia speciesand vaccines for inducing an immune response to Borrelia burgdorferi orother pathogenic Borrelia species. The proteins had not previously beenidentified or known to be antigens to which an immune response duringinfection is directed in humans or other animals. It is believed thatthe immunogenicity of recombinant forms of these proteins have not beenpreviously determined. An improved diagnostic test for Lyme disease isneeded and one or more of the proteins, by themselves or in combinationwith other recombinant proteins, should provide for better sensitivityand specificity than currently available assays. These proteins havealso not previously been investigated as sub-unit vaccines, either bythemselves or in combination with other recombinant proteins, forprotection against infection by Borrelia burgdorferi or other pathogenicBorrelia species. As such, in certain embodiments, the present inventionprovides vaccines using these proteins.

The encoding DNA sequences and the deduced proteins for BBK07, BBK12,and BBK19 were originally identified when the chromosome sequence andmost of the plasmid sequences for the B31 strain of Borrelia burgdorferi(Bb) were determined (Fraser et aI., Vature, 1997: Casjens et aI.,Alolecular Microbiology, 2000). They are located on the Ip36 linearplasmid of Bb. We have identified an orthologous DNA sequence to BBK07in another Borrelia species, Borrelia turicatae (Bt), a cause ofrelapsing fever. This DNA sequence and the deduced protein are not beenpublished or deposited in a public database.

The evidence of an orthologous gene in a distantly related species ofBorrelia as well as Bb indicates the genes for these proteins may occurin the other Borrelia species that cause Lyme disease. These include,but are not limited to, Borrelia afzelii (Ba) and Borrelia garinii (Bg).The chromosomes and the partial plasmid sequences for a single straineach of these species have been published but the deposited sequences donot show evidence of an ortholog of BBK07 and possible not the othergenes as well. We would expect to identify orthologs of BBK07, BBK12,and/or BBK 19 in Ba, Bg, and other agents of Lyme disease. In the caseof BBK07, this could be done by making an alignment of the Bb and Btortholog sequences and design polymerase chain reaction primers on thebasis of conserved sequence between the two genes. These primers wouldthen be used to amplify a part of the sought-after gene in these otherspecies. Once the sequence of the resultant cloned DNA was confirmed andcharacterized, we would use inverse PCR to amplify the 5′ and 3′ ends ofthe genes and thereby have the complete gene sequence. Alternatively wecould use the closed partial gene fragment as a probe for a DNA libraryof Ba or Bg in a plasmid, bacteriophage, or other cloning vector. Forthese methods, one could use low passage isolates of Ba and Bg obtaineddirectly from infected animals or to use field collected ticks that havebeen documented to contain either Sa or Bg. By the same approach, onecould also identify and isolate orthologs of BBK07, BSK12, and BBK19 inother relapsing fever species. including Borrelia hermsii.

On the other hand, if the existence of the putative orthologs in Baand/or Bg cannot be established, it indicates that one or more of thesegenes and their products are unique to Bb. In this case, a diagnostictest for Lyme disease that was based on detection of antibodies to oneor more the proteins would be specific for Bb. Such a test would be veryuseful in Europe and in Asia where the three species co-occur.Differentiating between infection with Bb or with one of the otherspecies is clinically important because infection with Bb is much morelikely to be associated with a chronic form of arthritis.

V. FliL (BB0279) and FlbB (BB0286)

In certain embodiments, the present invention comprises recombinantproteins of Lyme disease Borrelia species flagella-associated proteinsFliL and FlbB. In some embodiments, the methods are a diagnostic testfor antibodies to either or both FliL and FlbB in a variety of differentformats, in which the FliL and/or FlbB are alone or in combination withone or more other recombinant proteins. The diagnostic assay is forantibodies to Borrelia burgdorferi or another Lyme disease Borreliaspecies, such as B. afzelii and B. garinii. This assay may be used forlaboratory support of the diagnosis of Lyme disease, for staging theinfection, and for assessing the outcome of antibiotic therapy. Relatedproteins of relapsing Borrelia species, the syphilis agent Treponemapallidum, and the leptospirosis agent Leptospira interrogans could alsobe used as the basis for diagnostic assays for antibodies against theserespective etiologic agents.

We have experimental evidence of the immunogenicity and antigenicity ofFliL and FlbB in natural infections of Borrelia burgdorferi of humansand the wild mouse Peromyscus leucopus. These studies demonstrated thatassays based on one or both proteins were specific as well as sensitive.These data were obtained using an array of approximately 80% of the openreading frames of the Borrelia burgdorferi genome and sera from Lymedisease patients and controls and from infected and uninfected mice.

Two proteins of the flagellar apparatus of Borrelia burgdorferi andrelated Lyme disease (LD) agents, B. garinii and B. afzeiii, have beenidentified as important antigens for the serologic (i.e. antibody-based)diagnosis of LD. These are the FlaB protein, which is the majorflagellin of flagella and encoded by the flaB (open reading frame BBO147of the B. burgdorferi genome) gene, and FlgE, which is the hook proteinof the flagella apparatus and encoded by the flgE (B80283) gene. Therehave been several papers demonstrating the importance of the FlaB(formerly known as the “41 kDa” or “p41” protein) for serodiagnosis.

Purified flagella have also been reported as an antigen preparation fora serologic assay for Lyme disease and are the basis of at least onecommercial assay (Dako) for antibodies to LD Borrelia sp., and aflagella-based assay was used by the Centers for Disease Control for aperiod as a reference assay for LD diagnosis. These purified flagellawould contain FlaB and possibly FlgE but not the components of theexport mechanism, such as FIiL.

Since 1983 there have been several papers and other works that haveidentified antigens according to apparent molecular weight on SDSpolyacrylamide gels and Western blots. Included in this group is theFlaB (41 kDa) protein. Examples of other proteins that were firstrevealed as antigens through Western blots of native proteins were theOspA protein (“31 kDa”), BmpA protein (“39 kDa”), and Decorin-bindingProtein A (“18 kDa”). There have also several other proteins that havebeen identified as antigens of diagnostic importance when they wereexpressed as recombinant proteins in E. coli and then reacted with serafrom humans and other animals with infection with a LD Borrelia sp.Included in this group is the FlgE protein. While the fliL (BB0279) andflbB (BB0286) genes of B. burgdorferi and related species had beenidentified in sequence analysis of the parts or all of the genome andthe polypeptides encoded by the open reading frames deduced, we know ofno evidence that they were designated as informative antigens and ofdiagnostic importance previous to this disclosure. Neither is theirevidence that homologous FliL and FlbB proteins of other pathogenspirochetes, including the Borrelia species that cause relapsing fever,Treponema pallidum, the agent of syphilis, and Leptospira interrogans,the agent of leptospirosis, had been previously identified asinformative antigens of diagnostic importance for their respectivediseases. All three groups of organisms have sequences that arehomologous to the fliL gene of B. burgdorferi and other LD species. Thecalculated molecular weight of FIiL is 19929. There are multipleproteins migrating with this apparent size in SDS PAGE gels, and theycannot be distinguished. Although there is experimental evidence thatthe FIiL protein is expressed in vitro as well as in vivo, and thuswould be expected to present in the whole cell lysates in gels andWestern blots, the FliL protein may have not previously been recognizedas an important antigen because it is present in small amounts and alsobecause it would be predicted to migrate in the gel in an area with manyother proteins, which could not be discriminated.

VI. Kits

In yet other embodiments, the present invention provides kits for thedetection and characterization of Borrelia infection. In someembodiments, the kits contain antibodies specific for one or more of theantigens in Table 3, in addition to detection reagents and buffers. Inother embodiments, the kits contain reagents specific for the detectionof nucleic acid (e.g., oligonucleotide probes or primers). In someembodiments, the kits contain all of the components necessary and/orsufficient to perform a detection assay, including all controls,directions for performing assays, and any necessary software foranalysis and presentation of results.

Another embodiment of the present invention comprises a kit to test forthe presence of the polynucleotides or proteins. The kit can comprise,for example, an antibody for detection of a polypeptide or a probe fordetection of a polynucleotide. In addition, the kit can comprise areference or control sample; instructions for processing samples,performing the test and interpreting the results; and buffers and otherreagents necessary for performing the test. In other embodiments the kitcomprises pairs of primers (e.g., as shown in Table 4) for detectingexpression of one or more of the antigens in Table 3.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); μl(microliters); Xg (time gravity); and C (degrees Centigrade).

Example 1 Screening for Lyme Disease Related Antigens

This example describes genome-wide screening using arrays such that allor most open reading frames will be represented and screened withoutbias toward those expressed in greatest amounts in culture medium.

Materials and Methods

Bacterial strain, genome sequences, and primer design. Strain B31 of B.burgdorferi had undergone three passages since its isolation (6, 19).This organism was cultivated in BSK II broth medium (6). A high-passageisolate of strain B31 had been cloned by limiting dilution and had beenserially passed in culture medium at least 50 times. Whole-genome DNAwas extracted from the low-passage isolate as described previously (62).Primers were based on the sequences and annotations of the chromosomeand 21 plasmids of strain B31 (http://www., followed by“blackwellpublishing.com/products/journals/suppmat/mole/casjens.htm”)(21, 39). Forward and reverse primers were 20 nucleotides long and werecomplementary to the 5′ and 3′ ends of each ORF; peripherally they alsoincluded 33 nucleotide adapter sequences specific for plasmid pXT7 forrecombination cloning, as described previously (29). The forward andreverse primers for about 200 of the ORFs (that were identified asimmunogenic) are provided in Table 4 below:

TABLE 4 SEQ SEQ ID ID ORF 5′ Primer NO: 3′ Primer NO: BB_0056ATGTCAATAAAAACAGTAAA   1 ATTCTCCAAAACCTTAATAC   2 BB_0108ATGAAGAGTTTTTTATTTTG   3 TTTTAGACTAGAATCCAAGA   4 BB_0147ATGATTATCAATCATAATAC   5 TCTAAGCAATGACAAAACAT   6 BB_0181GTGGATTCAACATTTTCAGG   7 TACTCCCATTTTATTTATTA   8 BB_0215TTGATGAAAAAAGTTATTAT   9 TGTTTTTATCCCTAAAAAGC  10 BB_0238GTGTTAATGGAGGTTCTTAT  11 ATTTATGGAAGACAAAAACC  12 BB_0260TTGAAAGGGTTTTTAGCGAT  13 AACATATTGATCTTTTTCAT  14 BB_0279ATGCCTAATAAAGACGATGA  15 CATATCAAAAATATCAATTT  16 BB_0283ATGATGAGGTCTTTATATTC  17 ATTTTTCAATCTTACAAGTT  18 BB_0286ATGAAAGTGAATAATTTTTT  19 CTCCAATGAACTAACAGACA  20 BB_0323ATGAATATAAAGAATAAATT  21 TTTGGCAGGAATTATTATCT  22 BB_0328ATGAAATATATAAAAATAGC  23 TTTTTTAGTTTTAATATCTT  24 BB_0329ATGAAATTACAAAGGTCATT  25 TTTATTTTTTAATTTTAGCT  26 BB_0337ATGGGTTTTCACATTTATGA  27 TTTTTGTTTAATAGAATAAA  28 BB_0348ATGATTTCAAAGTTAACAAA  29 TATATTTCGTCCTTTGATTG  30 BB_0359ATGAAAAATAAATTTTTAAT  31 ATTACCTAATTTAGACAAAT  32 BB_0365ATGTATAAAAATGGTTTTTT  33 ATTCGTTAACATAGGTGAAA  34 BB_0385ATGTTAAAAAAAGTTTATTA  35 ATTTTCCATTTGCAAAACAA  36 BB_0408ATGTTTTTTAATTTTTTGAA  37 TTCAGATTCCTTTAATTTTA  38 BB_0476ATGAAATTTAGGAGGTTAGT  39 TTCCAATATCTCAAGAATTC  40 BB_0518ATGGGCAAAATAATAGGTAT  41 TTTTTTATCCTCGTCAACAA  42 BB_0543TTGTATATGATTAGGCTTAA  43 CACAATTCCAAATTCAAAAC  44 BB_0603ATGAAAAGCCATATTTTATA  45 GCTTCCGCTGTAGGCTATTT  46 BB_0649ATGGCTAAAGACATATATTT  47 CATCATTCCCATTCCTGGGT  48 BB_0652ATGAAAAAAGGATCTAAGCT  49 ATTACTCTTTGCATATTTTG  50 BB_0668TTGGTTTACATGAAAAGGAA  51 ATTTTTCGGAGATGATTCTT  52 BB_0681GTGGTTAGTATGAAGCTTAA  53 CTTTTCGATCTTAAAATAAT  54 BB_0751TTGGACTTGTTAGATTTACT  55 ATCTGCATTGTTGTGATGTT  56 BB_0772ATGAACAAACTAATGTTGAT  57 ATTTTGGTTTCCATCAATTT  58 BB_0774ATGATGAGAGCATTATGGAC  59 TTGCCTTTTTAAGTTATTTG  60 BB_0805TTGAGGAAAATATTAAAGTT  61 ATAATCTTTATCTCTAACAA  62 BB_0811ATGAAATACTTTTATTTTTT  63 ATAATCTTTTAAAAGCATTT  64 BB_0844ATGAAAAAAAAAAATTTATC  65 TTTACTCGTCTCTAAAAAAT  66 BB_A03TTGAAAAAAACGATTATTGT  67 TATAGTGTCTTTAAGTTTAT  68 BB_A04TTGAAAAGAGTCATTGTATC  69 GTTAATTAACGAATTAAATG  70 BB_A07GTGTGTGGGAGACGTATGAA  71 AAACGAAGCAGATGCATCAT  72 BB_A15ATGAAAAAATATTTATTGGG  73 TTTTAAAGCGTTTTTAATTT  74 BB_A16ATGAGATTATTAATAGGATT  75 TTTTAAAGCGTTTTTAAGCT  76 BB_A19ATGACGGCTTTACTTGAACG  77 CTTTTGTCTCTTTTTTATCC  78 BB_A25ATGAAAATTGGAAAGCTAAA  79 TTTCTTTTTTTTGCTTTTAT  80 BB_A34ATGATAATAAAAAAAAGAGG  81 TTCTTCTATAGGTTTTATTT  82 BB_A36TTGATGCAAAGGATAAGTAT  83 AACATTTCCATAATTTTTCA  84 BB_A40ATGAGCGATTCAATTGATTT  85 AATTGAATCTTTTATTTGCT  86 BB_A48ATGAGATACAAGTTAAAAAT  87 TTCATTGCTACCTTCTTGCA  88 BB_A57TTGAACGGCAAGCTTAGAAA  89 TTGATAATTTTTTTCTACCA  90 BB_A64TTGAAGGATAACATTTTGAA  91 CTGAATTGGAGCAAGAATAT  92 BB_A66TTGAAAATCAAACCATTAAT  93 CATTATACTAATGTATGCTT  94 BB_B09ATGAAATACCTTAAAAACAT  95 AAATTTATGCCTACTTGATT  96 BB_B14ATGATATTATATCAAAATCA  97 ACTTTTATAATCTTTATTTT  98 BB_B16ATGAAAATATTGATAAAAAA  99 TTTAATTGGTTTTATTTCAG 100 BB_B19ATGAAAAAGAATACATTAAG 101 AGGTTTTTTTGGACTTTCTG 102 BB_C03ATGGAAAAAAAACGTGTTGT 103 GATTTTTAGTTCTTCATATT 104 BB_C06ATGAGAAAAATAAGCCTATT 105 ATCTTTAGGCAAGTCTGCCA 106 BB_C10ATGCAAAAAATAAACATAGC 107 ATCTTCTTCAAGATATTTTA 108 BB_E09ATGCAAAAAGACATATATAT 109 TTCATCAATAAAAAGTTTTA 110 BB_F03TTGAGTATGGAACAACTAAT 111 CTTGAAATAGTTGCCAATTA 112 BB_G18ATGGCAGATTTCGATTTTAC 113 TGCAAATTTTCTGTTACCAT 114 BB_G33ATGAAATCATCAGTAGTGAC 115 TTTGAAATAATTGCTAATTA 116 BB_H06ATGAAAAAAAGTTTTTTATC 117 TAATAAAGTTTGCTTAATAG 118 BB_H13ATGAAAGCAGTTTTGGCAAC 119 TTTAAAAAATTTAGCAATTA 120 BB_I42ATGAGGATTTTGGTTGGCGT 121 TGTAGGTAAAATAGGAACTG 122 BB_J24ATGTTAAGGGCATTGTTAAT 123 GTAGTAGAAAGAATTGCCCT 124 BB_K07ATGAGTAAACTAATATTGGC 125 ATTATTAAAGCACAAATGTA 126 BB_K12ATGAGTAAACTAATATTGGC 127 GCTTAAAGTTGTCAATGTTT 128 BB K13TTGCTTTTAGGAGGTCAATC 129 ATCCAAATAATAAGAAACGG 130 BB_K19TTGAAAAAATATATTATCAA 131 ATTGTTAGGTTTTTCTTTTC 132 BB_K23ATGAAAGCCGTTATACCTAG 133 CTCAAATTTCAATCCCTTTG 134 BB_K32ATGAAAAAAGTTAAAAGTAA 135 GTACCAAACGCCATTCTTGT 136 BB_K52ATGAAAAAGAACATATATAT 137 TTCATCAGTAAAAAGTTTTA 138 BB_K53ATGAGGATTTTGGTTGGCGT 139 TGTAGGTAAAATAGAAACTG 140 BB_L03ATGAGTGATATAACAAAAAT 141 CCCTTTTATTGCTCTATTCC 142 BB_L27GTGTATAATATGACTATAAG 143 TTTGGAAATAAAAGCAAATA 144 BB_L39ATGGAGAAATTTATGAATAA 145 TTTTAAATTTCTTTTAAGCT 146 BB_L40ATGAATAAAAAAACAATTAT 147 ATCTTCTTCATCATAATTAT 148 BB_M27ATGAGAAATAAAAACATATT 149 ATTAGTGCCCTCTTCGAGGA 150 BB_M34ATGAACAATTTAGCATACAA 151 ATTTAAAAAATACTTATTGA 152 BB_M36ATGCTTATTAATAAAATAAA 153 CTTTAGTCTAAATATGCGCT 154 BB_N11TTGCCGCAAGATACAATTAG 155 AACTATATCTTGAGTAGTAA 156 BB_N27GTGTATAATATGACCATAAG 157 TTTAGAAATGAAAGCAAATA 158 BB_N28ATGAAAATTATCAACATATT 159 TTGCTGAGCTTGGCAGGTAC 160 BB_N34ATGAGAAATTTGGTGCACAG 161 ATTTAAAAAATGCTTATTGA 162 BB_N38ATGAATAAGAAAATGAAAAT 163 TTTTAAATTTTTTTTAAGCA 164 BB_N39ATGAATAAAAAAACATTGAT 165 CTGACTGTCACTGATGTATC 166 BB_O34ATGACTAATTTAGCGTACAG 167 ATTAAAGAAATACTTATTAA 168 BB_O39ATGAATAAGAAAATGAAAAT 169 TTCTTTTTTATCTTCTTCTA 170 BB_O40ATGAATAAAAAAATATTGAT 171 ATATGAATTACTATCCTCAA 172 BB_P34ATGACTAATTTAGCGTACAA 173 TTTGATATATTGTAAATATC 174 BB_P39ATGAATAAAAAAACAATTAT 175 ATCTTCTTCATCATAATTAT 176 BB_Q03ATGAGGATTTTGGTTGGCGT 177 TAAAATTTTTCCATTAATTG 178 BB_Q04ATGAAAAAGAACATATGTAT 179 TTCATCAGTAAAAAGTTTTA 180 BB_Q13TTGGGAGGATTTAATATGGA 181 ACTTTGTTTGATATGTACTT 182 BB_Q19GTGCTTAAAAGGGGGGCTAA 183 AGTGTTGTTTGGTTTAGTTT 184 BB_Q34ATGACCATAAGGGAAAATTT 185 TTTAGAAATGAAAGCAAATA 186 BB_Q35ATGAAAATCATCAACATATT 187 GTTTTGCCAATTAGCTGTAA 188 BB_Q40ATGGATAATAAAAAACCTAA 189 TTTAACATATTCATCATATA 190 BB_Q42ATGAATAGTTTGACTTACAG 191 TTTGCCACCTTGTAAATATT 192 BB_R12ATGCAATTTTATGATTTAAG 193 AGTGTTGTTTGGTTTAGATT 194 BB_R35ATGAGTAATTTAGCCTACAA 195 ATTGAAAAAACACTTATTAA 196 BB_R42ATGAATAAAAAAATAAAAAT 197 TTCTTTTTTACCTTCTACAG 198 BB_S30ATGAAAATCATCAACATATT 199 GCCACCATTATTGCAGTTAC 200 BB_S41ATGAATAAGAAAATGAAAAA 201 TTTTTTATCTTCTATATTTT 202Also included was the type K OspC protein gene of strain 297, inaddition to the type A OspC gene of B31 (12, 15). ORFs were namedaccording to the designations assigned to strain B31's genome (21, 39);“BB” followed by a four digit number (e.g., BB0279) indicates achromosome ORF, while “BB” followed by a third letter and a two-digitnumber (e.g., BBA25) indicates a linear or circular plasmid ORF, andeach replicon is assigned a separate letter (e.g., “A” for linearplasmid lp54 or “B” for circular plasmid cp26). As needed, genome ORFdesignations were supplemented with names in common use or whenpolypeptide identity has been inferred from homology to proteins withknown functions. The predictions of lipoproteins are those of Casjens etal. (http://www. followed by“blackwellpublishing.com/products/journals/suppmat/mole/casjens.htm”).

Array Production.

PCR amplification, cloning of amplicons into the plasmid vector, andthen transformation of E. coli DH5 were carried out as describedpreviously (29, 86). Of the 1,640 ORFs that were identified in the B.burgdorferi strain B31 genome (21, 39), 1,513 (861 chromosomal genes and652 plasmid genes) were subjected to PCR with the specific primers. Theremaining 127 ORFs had sequences that were so similar to the sequence ofat least one other ORF that PCR primers would not distinguish betweenthem. Of the 861 chromosomal ORFs that were attempted to be amplified,783 (91%) produced a product that was the correct size when PCR wasperformed, and 756 (88%) were successfully cloned into the vector. Ofthe 652 plasmid ORFs, 572 (88%) were amplified, and 536 (82%) werecloned into the plasmid vector. A sample consisting of 7% of 1,292clones from strain B31 was randomly selected for sequencing, and theinsert was confirmed in all cases. The coefficient of determination (R2)between the sizes of the ORFs and cloning success was only 0.05. Thefollowing 26 plasmid ORFs were randomly selected to be replicated on thearray: BBA03, BBA04, BBA14, BBA25, BBA52, BBA59, BBA62, BBA69, BBB07,BBB19, BBC06, BBJ50, BBK50, BBL28, BBL39, BBM38, BBN37, BBO40, BBP28,BBQ35, BBQ60, BBQ80, BBR28, BBR42, BBS30, and BBT07. As a negativecontrol, the arrays also contained 14 pairs of spots with the E. colicoupled transcription-translation reaction mixture without plasmid DNA.

Plasmid DNA was extracted and isolated using QlAprep spin kits (Qiagen).In vitro coupled transcription-translation reactions were performed withRTS 100 E. coli HY kits (Roche) in 0.2-ml tubes that were incubated for5 h at 30° C. The presence of the polyhistidine tag at the N terminus ofthe recombinant protein and the presence of the influenza Ahemagglutinin at the protein's C terminus were detected with monoclonalantibodies His-1 (Sigma) and 3F10 (Roche), respectively, and confirmedexpression in the in vitro reactions. Products oftranscription-translation reactions were printed in duplicate onnitrocellulose-coated glass slides (FAST; Whatman) using an Omni Grid100 apparatus (Genomic Solutions).

Protein Purification.

Plasmid DNA was extracted from selected clones and transformed in E.coli BL21 Star(DE3)/pLysS cells as described by the manufacturer(Invitrogen). The resultant transformants were cultivated in Terrificbroth (Bio 101 Systems) to stationary phase and, after harvesting bycentrifugation, were lysed with BugBuster buffer (Novagen). The lysatewas applied to a 5-ml HiTrap chelating HP affinity column (GEHealthcare). After the column was washed, bound proteins were elutedwith an imidazole step gradient using an Amersham Biosciences ÁKTA fastprotein liquid chromatography system operated with UNICORN 5.01software. The average amount recovered from a 1.0-liter culture was 1 to3 mg of protein with a purity of 80 to 90%, as estimated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis. Recovered proteinswere printed on array slides, as described above, or subjected topolyacrylamide gel electrophoresis with a 4 to 15% acrylamide gradientand then transferred to nitrocellulose membranes for Western blotanalysis (20). For printing on the array, the protein concentrationswere 0.03, 0.1, 0.3, and 0.9 mg/ml.

Serum Samples.

All serum samples were originally collected for other studies for whichinformed consent had been obtained; patient identifier information hadbeen removed. Serum panel 1 included samples from 48 adults collectedbetween 1990 and 1994, including 24 patients with erythema migrans(early infection), 19 patients with dissemination to other organs orother evidence of persistent infection (later infection), and 5 healthycontrols from a region where the organism is not endemic. These sampleswere provided by the Centers for Disease Control and Prevention, FortCollins, Colo., which had performed flagellin-based ELISA and IgG andIgM Western blot assays, as described previously (17, 50). Panel 1 alsoincluded sera from 13 healthy adult volunteers residing in California.

Serum panel 2 included serum specimens from 20 healthy adult controlsubjects, 20 adult patients with culture-positive erythema migrans(early infection), and 20 individuals with persistent LB witholiogoarticular arthritis (later infection). All 40 patients with Lymedisease met the criteria of the Centers for Disease Control andPrevention for diagnosis of Lyme disease (95). The 20 patients witherythema migrans were a random sample of 93 patients, seen in a study ofearly LB, from whom B. burgdorferi was cultured from erythema migransskin lesions (84, 93). Only convalescent samples, which were obtained atthe conclusion of 3 to 4 weeks of antibiotic therapy, were tested forthese patients, because seropositivity is more frequent duringconvalescence than during acute infection. The 20 patients with Lymearthritis were seen in 2006 and early 2007 in a study of susceptibilityto Lyme arthritis (82). For 10 of the 20 patients with Lyme arthritisthere was resolution of arthritis with antibiotic therapy(antibiotic-responsive arthritis), and for 10 there was not resolution(antibiotic-refractory arthritis). The samples were obtained when thepatients had active arthritis. All serum samples had been kept frozen at−80° C. until use.

Sera from 10 white-footed mice (P. leucopus), which had been capturedand then released after blood samples had been obtained at a field sitein Connecticut under an approved animal use protocol, were seropositiveas determined by a whole-cell ELISA and a Western blot assay, asdescribed previously (18). These sera were compared with sera from fouradult laboratory-reared P. leucopus mice that were obtained from thePeromyscus Stock Center, University of South Carolina, and wereseronegative as determined by the same assays.

Mouse Immunization.

Female, 4-week-old BALB/c mice (Jackson) were inoculatedintraperitoneally with 10 μg purified protein in phosphate-bufferedsaline (PBS) or PBS alone emulsified with Freund's complete adjuvant andwere boosted twice at 2-week intervals with the antigen solution or PBSalone in incomplete Freund's adjuvant. Plasma samples were collectedbefore each immunization and after the final boost using the MicrovetteCB 300 system for capillary blood collection (Sarstedt).

Antibody Reactions and Assays.

For experiments with arrays, human sera were diluted 1:200 in proteinarray blocking buffer (Whatman) that was supplemented with a lysate ofE. coli at a final protein concentration 5 mg/ml and then were incubatedat room temperature for 30 min with constant mixing (29). The arrayswere rehydrated in blocking buffer for 30 min, incubated with thepretreated sera for 12 h at 4° C. with constant agitation, washed in 10mM Tris (pH 8.0)-150 mM NaCl containing 0.05% Tween 20 buffer, and thenincubated with biotin-conjugated goat anti-human IgG(Fc—fragment-specific) serum (Jackson ImmunoResearch) that was diluted1:200 in blocking buffer. After the array slides were washed in 10 mMTris (pH 8.0)-150 mM NaCl, bound antibodies were detected withstreptavidin conjugated with the dye PBXL-3 (Martek). The washed andair-dried slides were scanned with a Perkin Elmer ScanArray Express HTapparatus at a wavelength of 670 nm and with an output of RGB formatTIFF files that were quantitated using ProScanArray Express software(PerkinElmer) with correction for spot-specific background. When P.leucopus serum was used, it was diluted 1:200 in protein blockingbuffer, and alkaline phosphatase-labeled goat anti-P. leucopus IgGantiserum (Kirkegaard and Perry) was used as the secondary antibody.Bound antibodies were detected using one-step nitrobluetetrazolium-5-bromo-4-chloro-3-indolylphosphate (BCIP) (Pierce). Arrayswere scanned at 2,400 dpi (Hewlett-Packard ScanJet 8200 scanner), andafter images were converted to gray scale format and inverted, they werequantitated as described above. Western blot analyses of whole lysatesof B. burgdorferi using 10 ng protein per lane or 250 ng purifiedprotein per lane were carried out as described previously (10).Nitrocellulose membranes were incubated with human or mouse serum at adilution of 1:250, and bound antibodies were detected by incubation withalkaline phosphatase-labeled goat anti-human IgG antiserum or anti-mouseIgG antiserum (Jackson ImmunoResearch) at a dilution of 1:1,000. Themurine monoclonal IgG antibodies to OspA (BBA15) and FlaB (BB0147) usedwere H5332 and H9724, respectively (9, 10).

Data Analysis.

The raw values from array scans were the mean average intensities of allthe pixels in a pair of printed spots for each Orf or negative control;these raw values were then log transformed. A preliminary analysisshowed that there was no difference in interpretation whether or not themean value for DNA controls was subtracted from each raw value beforelog transformation, and, consequently, this additional step was notincluded.

The following analyses were carried out. (i) The mean and standarddeviation (SD) for each Orf with all control serum samples in each panelwere determined. For each Orf and for each serum sample, the number ofSDs above or below the mean for the control sera in the same serum panelfor the Orf in question was determined. For each sample all the Orfsthat had array spots with values that were 2 or 3 SDs above the mean forthe negative controls in the experiment for the given Orf were tabulatedand summed. The frequencies of each Orf that appeared in this cumulativelist were then determined. (ii) Bayesian microarray expression analysisand discriminatory antigen selection were performed with softwareadapted from Cyber-T for protein arrays (4, 86, 87). The correction ofHochberg and Benjamini was applied to control for false discoveriesunder the multiple test conditions (47). (iii) Cluster analyses wereperformed and graphic displays of array results were generated using theMultiExperiment Viewer v 4.0 software available from The Institute forGenomic Research (78). The Euclidian distance criterion with averagelinkage was used, and 1,000 bootstrap analyses with replacementiterations were carried out. (iv) Receiver operating characteristiccurves were generated for selected sets of Orfs using the packages“e1071” and “ROCR” in the R statistical environment, available at thehttp://www. followed by “R-project.org” website. (v) Standard asymptoticor exact statistical analyses of continuous data were carried out withthe SYSTAT version 11 (SYSTAT Software, Inc.) software, the StatExactversion 6 (Cytel Software Corporation) software, or Confidence IntervalAnalysis version 2.1.2 (2), available at the http://www. followed by“som.soton.ac.uk/cia” website. Unless noted otherwise, significancetests were two sided. For means, differences, and odds ratios (OR), 95%confidence intervals are indicated below.

Results

Proteome Array and Overall Binding of Antibodies.

The array comprised in vitro products of 1,292 ORFs of strain B31 and anadditional ospC allele from another strain for a total of 1,293 B.burgdorferi ORFs. In separate experiments array slides were incubatedwith samples of serum panels 1 and 2. A serum specimen from a patientwith early LB in panel 1 was replicated in the same experiment. ThePearson and Spearman correlation coefficients were 0.94 and 0.87,respectively, for paired log-transformed raw intensity values; the meanlog 10 difference between the replicates of this serum was only 0.07(95% confidence interval, 0.06 to 0.08) for the total set of 1,293 ORFs.For the replicates of the 26 Orfs on the array, the Pearson and Spearmancorrelation coefficients were 0.84 and 0.84, respectively, for panel 1and 0.81 and 0.83, respectively, for panel 2. The corresponding meanlog_(in) differences were 0.07 (95% confidence interval, 0.00 to 0.15)and 0.04 (95% confidence interval, −0.03 to 0.11) for the two panels.

FIG. 1 provides an overall summary of the data obtained by pairwiseplotting of log-transformed values for each ORF for the three clinicalgroups of each serum panel (controls, early infection, and laterinfection). This figure includes the distributions for each group as awhole, as well as medians for the distributions and the results of anonparametric analysis of the paired data. With infection and itsadvance, medians shifted slightly to the right in the distributions ofaverage intensities per ORF by clinical group. More notable were thelonger right-handed tails of the distributions, which are also reflectedby the several outlying points in the scatter plots for patient seraversus controls and for late-infection sera versus early-infection sera.The two panels differed in whether early-infection sera could bedistinguished from late-infection sera by a nonparametric statistic(FIG. 1).

The analysis described above first averaged ORF values across clinicalgroups and then examined correlations for each of the 1,293 ORFs. If,instead, average ORF intensities by serum sample for all 1,293 ORFs werecalculated first and then the averages by serum were compared byclinical stage, heterogeneity of the results for individual sera wasobserved as overlapping distributions for controls and patient groups.For the first serum panel the mean differences in raw intensity valuesbetween patient sera and controls were 42 (95% confidence interval, −135to 218) for early infection and 157 (95% confidence interval, −3 to 318)for later infection; the corresponding differences from controls forpanel 2 were −36 (95% confidence interval, −220 to 149) for earlyinfection and 8 (95% confidence interval, −185 to 202) for patients withLyme arthritis. Thus, the total amount of antibody binding to the array,which is analogous to a whole-cell assay, could not be used to assignsera to infection and control bins with confidence. More promising forthis purpose was the smaller number of ORFs populating the long tail inthe distributions and the “outliers” in the plots shown in FIG. 1.

By using pairwise comparisons of all sera for individual ORFs, it wasestimated that the upper of limit of the number of strain B31 Orfs thatwere informative as immunogens was 200 of the 1,292 Orfs on the array(see below). To identify these immunogens, two complementary approacheswere used. The first approach was based on an often-used criterion forsetting a “cutoff” between interpretations of positive and negative forserological assays, namely, values that were 3 SDs above the average forcontrol sera in the same run. Before using this approach, it was firstdetermined whether variances were out-of-proportion high when the meanvalues for control specimens increased. This would have been reflectedin a significant increase in the coefficient of variation (CV) (i.e., SDdivided by the mean) as the mean increased. For the 1,292 B31 Orfs andthe panel 1 control sera, the mean CV was 0.115 (95% confidenceinterval, 0.113 to 0.117), and there was little correlation (R=0.06)between the mean and the CV over the range of means. Inasmuch as the SDsfor the control sera were the same in both experiments (namely, 0.85 forthe 18 control sera in panel 1 and 0.84 for the 20 control sera in panel2), normalizing the data in units of SDs allowed the data sets to becombined for the two panels. Using a simulation procedure (see below),we found that for the combined set of the later LB panel 1 and 2 sera,ORF values that exceeded the cutoff at a frequency of 6 or more times,out of a possible 39, were unlikely by chance at a one-tailed level ofconfidence of 0.025.

The log-normalized data for later LB sera and controls of both panelswere also examined using a Bayesian statistical procedure (4), usingsoftware originally developed for DNA microarray analysis and thenmodified for antibody binding to proteome arrays (86, 87). For each ORF,an analysis of variance (ANOVA) comparing control sera with later LBsera was performed. In this analysis, the empirical sample variances arereplaced with Bayes-regularized variance estimates. TheBayes-regularized variance is obtained by incorporating both theempirical sample variance and the variance of proteins with similarintensity levels (3, 4). This analysis produced F scores and P valuesthat were used to rank the ORFs. The log_(in) of the F score correlatedwith the frequencies based on a cutoff of 3 SDs (R2=0.73 as determinedby linear regression).

Identification of immunogens. The most informative of the 200immunogenic Orfs were then identified. Table 1 lists in alphabetical andnumerical order the 84 Orfs whose values were 3 SDs above the controlvalues 6 or more times out of a possible 39, had F scores of >11, andhad corrected P values of <0.001. The Orfs with the highest frequenciesof values that were 3 SDs above the control values were BBG33, BB0279,BBL27, and BBA25. An additional 19 Orfs, for a total of 103 (8.0%) outof 1,292 Orfs, had P values of <5×10-4 as determined by theBayes-regularized analysis. This additional group included VlsE (BBF33)and the chaperonin GroEL (BB0649). The mean numbers of amino acids were293 (95% confidence interval, 266 to 320) for the 103 Orfs on the list,compared to 260 (95% confidence interval, 248 to 272) for the other1,190 Orfs (P=0.11, t test). Thus, immunogenicity was not associatedwith length of the protein. Moreover, there was no difference betweenthe two groups of Orfs in terms of the amount of protein on the array,as measured by the raw values for antibody binding to the hemagglutininmoiety of the recombinant proteins; the values were 2,816 (95%confidence interval, 2,369 to 3,349) for the 103 Orfs and 2,618 (95%confidence interval, 2,493 to 2,750) for the other Orfs (P=0.42).

TABLE 1 Immunogenic proteins of B. burgdorferi in natural infections ofhumans and mice Later Early Lyme Lyme Predicted disease F P disease Miceto be ORF^(a) (n = 39)^(b) score^(c) value^(d) (n = 44)^(e) (n = 10)^(f)PF lipoprotein^(g) Deduced gene product^(h) BB0056 12 18.7 6.0E−04 4 (5)0 − Phosphoglycerate kinase BB0108 9 18.1 7.9E−04 1 (2) 0 −Peptidylprolyl isomerase BB0147 17 80.7 4.8E−12  5 (17) 4 − Flagellarfilament (FlaB) BB0181 2 27.1 2.6E−05 1 (3) 0 − Flagellarhook-associated protein (FlgK) BB0215 10 55.3 3.3E−09 2 (7) 3 +Phosphate ABC transporter (PstS) BB0238 7 33.8 2.5E−06 2 (3) 0 −Hypothetical protein BB0260 5 19 5.4E−04 3 (4) 2 − Hypothetical proteinBB0279 34 246.9 0 10 (16) 7 − Flagellar protein (FliL) BB0283 32 149.4 02 (3) 1 − Flagellar hook protein (FlgE) BB0286 0 29.4 1.2E−05 0 (1) 7 −Flagellar protein (FlbB) BB0323 9 39.8 3.3E−07 1 (2) 2 + Hypotheticalprotein BB0328 2 27 2.7E−05 0 (1) 0 37 + Oligopeptide ABC transporter(OppA-1) BB0329 30 192.2 0 7 (9) 0 37 + Oligopeptide ABC transporter(OppA-2) BB0337 5 17 1.2E−03 0 (0) 0 − Enolase BB0348 14 59.5 1.1E−09 1(6) 1 − Pyruvate kinase BB0359 15 50.3 1.3E−08 1 (2) 0 −Carboxy-terminal protease BB0365 15 43.8 9.9E−08 4 (8) 4 + LipoproteinLA7 BB0385 6 29.3 1.2E−05 2 (3) 2 + Basic membrane protein D (BmpD)BB0408 16 32 4.7E−06 0 (4) 4 − Phosphotransferase system,fructose-specific IIABC BB0476 7 19.9 3.9E−04 0 (2) 1 − Translationelongation factor TU (Tuf) BB0518 17 62.7 4.7E−10 3 (5) 2 − Molecularchaperone (DnaK) BB0543 7 45.2 6.4E−08 2 (3) 2 − Hypothetical proteinBB0603 11 54.7 3.9E−09  3 (10) 3 − P66 outer membrane protein BB0649 120.8 2.7E−04 0 (3) 1 − Chaperonin (GroEL) BB0652 5 16.6 1.4E−03 2 (2) 0− Protein export protein (SecD) BB0668 6 21.7 1.9E−04 1 (3) 1 −Flagellar filament outer layer protein (FlaA) BB0681 8 36.2 1.1E−06 1(4) 2 − Methyl-accepting chemotaxis protein BB0751 6 24.6 6.5E−05 0 (1)1 − Hypothetical protein BB0772 6 14.8 2.9E−03 1 (2) 0 − FlagellarP-ring protein (FlgI) BB0774 11 44.6 7.8E−08 1 (1) 2 − Flagellar basalbody cord protein (FlgG) BB0805 6 13.2 5.5E−03 0 (2) 1 −Polyribonucleotidyltransferase (PnpA) BB0811 9 20.4 3.1E−04 2 (4) 0 −Hypothetical protein (COG1413) BB0844 7 43.1 1.2E−07 0 (2) 10 12 +Hypothetical protein BBA03 13 35.3 1.5E−06 1 (4) 3 + Hypotheticalprotein BBA04 7 12.5 7.1E−03 0 (1) 3 44 + “S2 antigen” BBA07 17 40.62.7E−07 1 (4) 0 + Hypothetical protein BBA15 16 28.3 1.7E−05 3 (6) 253 + Outer surface protein A BBA16 22 58 1.6E−09 1 (2) 0 53 + Outersurface protein B BBA19 1 22 1.7E−04 1 (3) 3 50 − Hypothetical proteinBBA25 33 134.5 0 23 (27) 10 74 + Decorin binding protein B BBA34 13 76.61.3E−11 1 (2) 0 37 + Oligopeptide ABC transporter (OppA-5) BBA36 20 52.28.0E−09 5 (7) 7 + Hypothetical protein BBA40 10 17.2 1.1E−03 1 (1) 1 148− Hypothetical protein BBA48 5 33.4 2.8E−06 1 (3) 0 154 − Hypotheticalprotein BBA57 9 39.9 3.3E−07 2 (6) 9 + Hypothetical protein BBA64 1475.6 1.6E−11  6 (14) 7 54 + Hypothetical protein BBA66 7 34.4 2.0E−06 4(6) 0 54 + Hypothetical protein BBB09 14 34.7 1.9E−06 0 (0) 2 +Hypothetical protein BBB14 13 60.9 7.3E−10 1 (1) 0 + Hypotheticalprotein BBB16 8 37.1 8.3E−07 0 (2) 0 37 + Oligopeptide ABC transporter(OppA-4) BBB19-A 21 79.5 6.3E−12 23 (26) 10 + OspC type A (strain B31)BBB19-K 24 54.1 4.5E−09 16 (21) 7 + OspC type K (strain 297) BBC03 515.1 2.6E−03 1 (4) 0 49 − Hypothetical protein BBC06 8 28.3 1.7E−05 1(2) 1 95 − EppA (BapA) BBC10 11 15.7 2.0E−03 0 (0) 6 63 + RevA BBE09 417 1.2E−03 1 (2) 4 44 + Hypothetical protein BBF03 23 69.1 8.3E−11 1 (3)0 80 − BdrS (BdrF1) BBF33 3 140 0  3 (21) 9 + VlsE BBG18 7 28.7 1.5E−051 (2) 0 − Hypothetical protein BBG33 36 286.2 0 11 (16) 10 80 − BdrT(BdrF2) BBH06 16 57.1 2.1E−09 2 (2) 0 + Hypothetical protein BBH13 30140.7 0 5 (8) 4 80 − BdrU (BdrF3) BBI42 16 61.5 6.4E−10 1 (1) 0 52 +Hypothetical protein BBJ24 6 45.5 6.0E−08 0 (1) 0 106 − Hypotheticalprotein BBK07 13 40 3.3E−07 11 (21) 10 59 + Hypothetical protein BBK1218 41.8 1.8E−07 12 (22) 9 59 + Hypothetical protein BBK13 8 25.5 4.7E−050 (3) 2 40 − Hypothetical protein (COG2859) BBK19 12 67.8 1.2E−10 4 (6)8 + Hypothetical protein BBK23 4 19.5 4.6E−04 2 (4) 1 62 − Hypotheticalprotein BBK32 22 122.3 0 13 (17) 9 + Fibronectin-binding protein BBK52 325.4 4.9E−05 1 (3) 4 44 + “P23” BBK53 10 31.5 5.5E−06 0 (3) 3 52 +Hypothetical protein BBL03 9 23.7 9.0E−05  6 (11) 0 148 − Hypotheticalprotein BBL27 33 229.6 0  6 (11) 4 80 − BdrO (BdrE1) BBL39 5 23.88.9E−05 0 (3) 4 162 + ErpN (CRASP-5) BBL40 22 51.5 9.4E−09 6 (7) 10163 + ErpO BBM27 18 51.6 9.3E−09 2 (5) 6 63 + RevA BBM34 27 153.1 0 3(7) 6 80 − BdrK (BdrD2) BBM36 3 21.5 2.1E−04 0 (2) 0 144 − Hypotheticalprotein BBN11 23 84.9 1.8E−12 2 (3) 0 152 − Hypothetical protein BBN2727 143.9 0 3 (6) 5 80 − BdrR (BdrE2) BBN28 6 21 2.5E−04 0 (2) 0 113 +MlpI BBN34 31 192.1 0  8 (13) 4 80 − BdrQ (BdrD10) BBN38 20 56.2 2.6E−097 (9) 2 162 + ErpP (CRASP-3) BBN39 23 42.2 1.6E−07 5 (9) 9 163 + ErpQBBO34 27 122.7 0 4 (6) 3 80 − BdrM (BdrD3) BBO39 23 71.1 5.0E−11 2 (6) 8164 + ErpL BBO40 6 23.4 1.0E−04 0 (2) 6 164 + ErpM BBP34 31 190.9 0  6(11) 3 80 − BdrA (BdrD4) BBP39 12 25.6 4.6E−05 4 (9) 9 163 + ErpB BBQ0327 101.9 3.4E−14 1 (2) 5 52 + Hypothetical protein BBQ04 6 23.3 1.1E−041 (3) 4 44 + Hypothetical protein BBQ13 1 15.7 2.0E−03 1 (1) 2 149 −Hypothetical protein BBQ19 6 16.4 1.5E−03 3 (5) 2 153 − Hypotheticalprotein BBQ34 30 170.4 0 3 (7) 7 80 − BdrW (BdrE6) BBQ35 3 15.7 2.0E−031 (3) 3 113 + MlpJ BBQ40 6 11.7 9.8E−03 3 (5) 0 32 − Partition proteinBBQ42 30 179.6 0 6 (9) 1 80 − BdrV (BdrD5) BBR12 6 13.3 5.3E−03 0 (2) 1153 − Hypothetical protein BBR35 8 30.1 9.2E−06 1 (3) 0 80 − BdrG BBR4214 47.3 3.4E−08 1 (1) 5 164 + ErpY BBS30 0 20.5 3.0E−04 0 (4) 4 113 +MlpC BBS41 18 29.8 1.0E−05 1 (3) 7 164 + ErpG ^(a)Bold type indicates anORF that had a P value of <0.005 but whose frequency for later Lymedisease sera was <6. ^(b)The numbers are the numbers of serum sampleswhose values were ≧3 SDs above the mean of the controls for the panel. nis the number of individuals in the group for combined panel 1 and 2sera. ^(c)The F score is the Bayes-regularized variance (see the text).^(d)The P value is the corrected P value (0, P < 1.0E−14). ^(e)Thenumbers are the numbers of LB patient serum samples whose values were ≧3SDs or ≧2 SDs above mean of the controls for panels 1 and 2. n is thenumber of individuals in the group for combined panel 1 and 2 sera.^(f)The numbers are the numbers of P. leucopus sera (out of 10) whosevalues are ≧3 SDs above the mean for four control P. leucopus mice.^(g)+, protein predicted to be a lipoprotein; −, protein not predictedto be a lipoprotein. ^(h)Alternative protein designations are given inparentheses.

Several proteins that were known antigens and valuable for serodiagnosiswere on the list. These proteins included FlaB (BB0147) (9, 45), the P66outer membrane protein (BB0603) (5, 16), OspA and OspB (BBA15 and BBA16)(48), decorin-binding protein B (BBA25) (37, 46), OspC (BBB19) (68, 96),fibronectin-binding protein (BBK32) (71), and VlsE (BBF33) (54, 56). Theother reactive Orfs that were previously reported to elicit antibodiesduring infections of humans or experimental animals were as follows: LA7(BB0365) (53, 94), the chaperonins DnaK (BB0518) and GroEL (BB0649)(58), FlgE (BB0283) (51), some Erp proteins (59, 85), oligopeptide ABCtransporters (OppA; BB0328, BB0329, BBA34, and BBB16) (25, 28, 65), “S2antigen” (BBA04) (36), the paralogous BBA64 and BBA66 proteins (65),RevA proteins (BBC10 and BBM27) (41, 65), EppA/BapA (BBC06) (63), Mlpproteins (BBN28, BBQ35, and BBS30) (70), and some Bdr proteins (99).

There were several Orfs that previously either were not recognized asimmunogens during infection or had received little attention. Notableamong this group were the following: (i) the paralogous BBK07 and BBK12lipoproteins; (ii) BBK19 and BBK53, two other lipoproteins encoded byplasmid lp36; (iii) several more flagellar apparatus proteins, includingFliL (BB0279), FlaA (BB0668), and FlgG (BB0774); (iv) additionalparalogous family (PF) 44 proteins (BBE09, BBK53, and BBQ04); (v)BB0260, BB0323, BB0543, and BB0751, hypothetical proteins encoded on thechromosome; (vi) BBA03, BBA07, BBA36, and BBA57, hypothetical proteinsor lipoproteins uniquely encoded by lp54; and (vii) BBG18 and BBH06,unique hypothetical proteins encoded by other plasmids. On the list ofnew immunogens there were only a few chromosome-encoded Orfs that werehomologous to proteins having established functions in other bacteria,such as the phosphate ABC transporter PstS (BB0215), pyruvate kinase(BB0348), a carboxy-terminal protease (BB0359, and a methyl-acceptingchemotaxis protein (BB0681).

Whereas plasmid-encoded Orfs accounted for 536 (41%) of the 1,292 B31Orfs on the array, 70 (69%) of the 102 immunogenic Orfs of strain B31are plasmid encoded (OR, 3.1 (95% confidence interval, 2.0 to 4.9);exact P<10⁻⁶). Fifty-nine (58%) Orfs, all but two of which were plasmidencoded, belonged to 1 of 26 PFs. Of a possible 174 Orfs that belong to1 of these 26 PFs, 114 (66%) were included as amplicons on the array.The greatest representation was that of PF 80, which comprises the Bdrproteins; 12 (92%) of a possible 13 Orfs were on the list of 83 Orfs.These Orfs included high-ranking BBG33 and BBL27 proteins. Other PFswith three or more representatives on the list were the PFs containingthe Erp proteins (PFs 162 to 164), oligopeptide ABC transporters (PF37), Mlp proteins (PF 113), the “S2 antigen” and related proteins (PF44), and a set of hypothetical proteins with unknown functions (PF 52).

For tabulation of the plasmid locations of the ORFs shown in Table 1,pseudogenes and ORFs were excluded that were less than 300 nucleotideslong (21). The sizes of linear plasmids lp38 (38,829 nucleotides) andlp36 (36,849 nucleotides) are similar. Only 1 of lp38's 17 ORFs, BBJ24,was among the ORFs encoding high-ranking antigens, but 8 of the 19 lp36ORFs were (OR, 11.6 (95% confidence interval, 1.2 to 548); P=0.03). Thepresence of plasmid lp36 has been associated in one study withinfectivity or virulence in a mammalian host (49), as has been thepresence of lp25 in another study (72), but only BBE09 of the 10 ORFs oflp25 were among the ORFs encoding immunogens.

Forty-eight (48%) of the 102 immunogens of strain B31 are lipoproteinsas determined by prediction or empirical documentation. Of the 756chromosome-encoded Orfs included in the array, only 32 (4%) arelipoproteins, but, as shown in Table 1, 7 (21%) of the 33chromosome-encoded Orfs among the immunogens are lipoproteins (OR, 6.1(95% confidence interval, 2.1 to 15.8); P=0.001). Whereas 85 (16%) ofthe 536 plasmid-encoded Orfs on the array are predicted lipoproteins, 41(59%) of the 70 plasmid-encoded proteins of strain B31 on the antigenlist are predicted lipoproteins (OR, 7.6 (95% confidence interval, 4.3to 13.4); P<10⁻¹²). In addition to five documented outer membraneproteins (OspA, OspB, OspC, VlsE, and P66), the following threehypothetical proteins among the immunogens were predicted to localize tothe outer membrane by the PSORT algorithm for double-membrane bacteria(40): BB0260, BB0751, and BB0811.

Stage of Infection.

In general, sera from early in infection reacted with fewer antigens perserum sample and antigens from a narrower list of antigens. For 20 (83%)of the 24 panel 1 early LB cases there was at least one Orf in Table 1whose value exceeded the 3-SD cutoff. Of the four cases for which therewas not at least one Orf whose value exceeded the 3-SD cutoff, three(75%) were seronegative as determined by ELISA and IgG and IgM Westernblotting. Of the 20 cases of early infection for serum panel 2, 17 (85%)had at least one Orf whose value was SDs. For the 37 samples with one ormore reactive Orfs, the number of Orfs whose values were above thethreshold ranged from 1 to 37, and the median number was five Orfs persample. For the 84 antigens identified by the first analysis, the valuesfor 69 (82%) were above the threshold for at least one of theearly-infection sera (Table 1). In most cases, the following 15 Orfswhose values fell below the cutoff with all early sera were also amongthe least prevalent Orfs for sera obtained later in disease: BB0408,BB0476, BB0751, BB0805, BB0844, BBA04, BBB09, BBB16, BBC10, BBJ24,BBK13, BBK53, BBN28, BB040, and BBR12. The Orfs whose values exceededthe cutoff in at least 10 of the 37 samples were, in descending order,BBA25 (DbpB), BBB19 (OspC type A), BBB19 (OspC type K), BBK32(fibronectin-binding protein), BBK12, BBG33 (BdrT), BBK07, and BB0279(FliL).

Sera of the 10 patients with refractory Lyme arthritis were comparedwith sera of the 10 patients with arthritis that responded to antibiotictherapy. As determined by a t test and nonparametric rank test oflog-transformed values, there was not a significant difference (P>0.05)between the two groups for any of the 1,293 Orfs, including both OspCproteins.

White-footed mouse antibodies. Using the same batch of genome-widearrays, the reactions of sera from 10 P. leucopus mice were examinedthat were captured in an area in which the level of B. burgdorferiinfection of mice approached 100% by the end of the transmission season(18). All 10 mice were seropositive as determined by the whole-cellassay and Western blot analysis (18). These sera were compared with serafrom four laboratory-reared P. leucopus mice. As described above, thenumber of SDs above or below the mean of the controls was calculated foreach Orf and each mouse serum. Of the 103 Orfs shown in Table 1, only 30(29%) were not represented at least once among the Orfs with values ofSDs with P. leucopus sera. The highest frequencies (≧7 of 10 sera) werethose of the following Orfs, in alphabetical order: BB0279, BB0286,BB0844, BBA25, BBA36, BBA57, BBA64, BBB19 (OspC types A and K), BBF33,BBG33, BBK07, BBK12, BBK19, BBK32, BBL40, BBN39, BBO39, BBP39, BBQ34,and BBS41. Thirteen Orfs had frequencies of 5 among the 10 P. leucopussera but were not among the high-ranking Orfs with human sera (Table 1).These Orfs included two hypothetical proteins (BB0039 and BB0428), twomembers of PF 143 (BBP26 and BBS26), and the BBK50 protein, anotherlp36-encoded protein. But also represented among the high-ranking Orfswith P. leucopus sera were members of PFs, at least one of which wasfrequently recognized by human antibodies, including two additional PF113 proteins, M1 pH (BBL28) and M1 pA (BBP28); another PF 164 protein,ErpK (BBM38); and an additional PF 54 protein, BBA73. Overall, there wasconsiderable overlap in the sets of immunogens for humans and P.leucopus infected with B. burgdorferi.

Second Array.

To confirm the results described above, we produced a second array with66 recombinant proteins selected from the 103 Orfs shown in Table 1. Thesecond array contained three additional proteins that were not clonedfor the first array. Two of these, BB0383 (BmpA or P39 protein) andBB0744 (P83/100 protein), are among the 10 signal antigens for acommonly used criterion for Western blot interpretation (33). The thirdadditional ORF was BBA24 or decorin-binding protein A (DbpA). Thesmaller arrays were incubated with 12 later LB sera and three controlsera from panel 1. FIG. 2 shows the results in a two-color gradientformat with an accompanying cluster analysis. BB0383 and BB0744clustered with several other proteins that were frequently bound byantibodies of LB sera, including FlaB (BB0147), BB0279 (FliL), VlsE, andDbpB (BBA25). The patterns of reactivity with different patient andcontrol sera were essentially the same for these antigens. This wasdemonstrated by correlations between the Orfs; for BB0383, the R² valuesfor BB0279, BB0147, VlsE, and BBA25 were 0.90, 0.86, 0.70, and 0.58,respectively, and for BB0744, the corresponding R² values were 0.91,0.81, 0.71, and 0.74, respectively. DbpA (BBA24), whose sequence isgenetically more diverse than that of DbpB across strains (76), was lessfrequently reactive with the collection of patient sera than DbpB. Thus,addition of the P83/100, BmpA, or DbpA protein to the array providedlittle or no additional discriminatory power.

FIG. 2 also shows clustering of the Bdr proteins and the BBK07/BBK12proteins but not of the two OspC proteins in terms of their patterns andintensities of reactivities with this set of sera. The relationshipbetween clustering of Orfs and amino acid sequence identity was examinedby plotting normalized values for individual panel 1 control, early LB,and later LB sera and for selected pairs of Orfs (FIG. 3). There washigh correlation between the paralogous proteins BBK07 and BBK12 andbetween two Bdr proteins, BBG33 and BBL27, in the serum antibodyreactions. The data corresponded to amino acid sequence identities of 87and 80%, respectively. In contrast, there were greater differences inthe reactivities of sera with the two OspC proteins, even though theoverall level of identity between them was close to that of the two Bdrproteins. Lower still was the correlation between two high-rankingproteins which are similar sizes but are not homologous, BBK07 andBBA25.

Purified Proteins.

Five Orfs were selected for further investigation as purifiedrecombinant proteins: BB0279 (FliL), (FlgE), BBA25 (DbpB), BBG33 (BdrT),and BBK12. Western blot analyses were carried out with sera from 17patients with later LB and five panel 1 controls (FIG. 4). Binding thatwas noted in the array was confirmed by Western blotting; no bands wereobserved with the control sera. Different amouts of proteins BBA25,BBG33, BBK12, and BB283 were then used over a 30-fold range in an arrayformat and incubated the chips with the same patient and control sera(FIG. 5). While for some proteins the binding by control sera increasedwith higher protein concentrations, the log-transformed raw values forpatient sera changed little over the concentration range used, anindication that the absolute amount of protein in the spots of thehigh-throughput array over this range was not a major determinant of theamount of antibody binding as estimated by digitization of the signalsfor this study. When binding to in vitro-produced proteins on the arraywas compared to binding to different amounts of a purified protein for agiven Orf for a standard curve, we estimated that the amounts of Orfs inthe genome-wide array were 50 to 400 pg protein per spot.

While detection of antibody to an Orf was evidence of expression of thepredicted polypeptide, this evidence was indirect. One of the purfiedproteins, BBK12, was used to immunize mice and thereby provide a reagentfor more direct documentation of expression. It is noted that suchimmunization could be performed with any of the proteins found to beimmunogenic (e.g., in Table 1 or Table 3) in order to generated anantibody reagent for diagnostic or other applications. This Orf waschosen because it and the product of a paralogous gene, BBK07, had notbeen previously reported to be immunogenic. In fact, there was littleprevious comment on either of these proteins beyond their annotation ashypothetical lipoproteins with unknown functions. FIG. 6 shows a Westernblot in which lysates of low-passage or high-passage strain B31 wereincubated with the anti-BBK12 antiserum or monoclonal antibodies to OspA(BBA15) or FlaB (BB0147). Because of the probable antigeniccross-reactivity between BBK07 and BBK12, as the analysis shown in FIG.3 suggests, it could not be assumed that the antiserum could easilydistinguish between the two Orfs. As expected, FlaB and OspA wereexpressed by both low- and high-passage isolates of strain B31. Incontrast, the BBK12 and/or BBK07 protein was detected in low-passagecells but not in high-passage cells. This experiment not only confirmedthat there was expression of either BBK12 or BBK07 or both but alsoshowed that loss of expression of these proteins was associated withhigh passage. Thus, one explanation for the previous lack of recognitionof informative antigens, such as BBK07 and BBK12, was thathigher-passage cell populations, which were often used as the basis ofdiagnostic assays, did not express the proteins, either because ofplasmid loss or because of transcriptional or translational failure.

How Many Antigens are Sufficient?

This Example permitted study an estimation of the minimum number ofantigens that would be needed to achieve a highly specific B.burgdorferi diagnostic assay. For this, the discriminatory power ofdifferent sets of ORFs was studied using receiver operatingcharacteristic (ROC) curves, where the false-positive rate(1—specificity) is the x axis and the true positive rate (sensitivity)is they axis for different thresholds of the underlying classifier. Thearea under the curve (AUC) summarizes the results. An AUC of 1.0indicates a perfect classifier, while an AUC of 0.51 (95% confidenceinterval, 0.38 to 0.64) is the expected value for a classifier thatworks by chance for the data set, as inferred by the method of Truchonand Bayly (89). The log-transformed data for controls and later LB serafrom both panels were used for this analysis. First, ROC curves weregenerated for single antigens to assess the ability to separate thecontrol and disease. The Orf number is the rank based on theBayes-regularized ANOVA F score (see Table 5).

TABLE 5 Open reading Controls Mean area Lower Upper Rank frame meanunder curve 95% CI 95% CI 1 BBG33 0.021 0.988 0.985 0.991 2 BB0279 0.0300.978 0.973 0.983 3 BBL27 −0.034 0.997 0.997 0.998 4 BB0329 0.026 0.9810.976 0.986 5 BBN34 0.020 0.984 0.981 0.988 6 BBP34 0.067 0.987 0.9850.990 7 BBQ42 0.000 0.984 0.981 0.988 8 BBQ34 −0.022 0.980 0.976 0.985 9BBM34 0.023 0.977 0.973 0.980 10 BB0283 −0.035 0.949 0.944 0.953 11BBN27 −0.016 0.981 0.977 0.985 12 BBH13 0.019 0.958 0.953 0.963 13 VlsE−0.012 0.968 0.963 0.972 14 BBA25 0.008 0.974 0.968 0.981 15 BBO34 0.0390.965 0.961 0.969 16 BBK32 −0.022 0.974 0.970 0.979 17 BBQ03 0.003 0.9260.917 0.934 18 BBN11 0.018 0.951 0.947 0.955 19 BB0147 0.022 0.967 0.9610.973 20 OspC_A 0.019 0.911 0.904 0.919 21 BBA34 0.025 0.967 0.963 0.97122 BBA64 0.024 0.927 0.917 0.937 23 BBO39 0.000 0.905 0.897 0.913 24BBF03 0.028 0.903 0.897 0.909 25 BBK19 0.016 0.897 0.885 0.909 26 BB0518−0.014 0.933 0.928 0.938 27 BBI42 0.016 0.928 0.921 0.935 28 BBB14 0.0300.917 0.911 0.924 29 BB0348 −0.003 0.896 0.887 0.905 30 BBA16 0.0160.910 0.905 0.915 31 BBH06 −0.001 0.898 0.889 0.907 32 BBN38 0.007 0.8810.869 0.892 33 BB0215 0.021 0.885 0.877 0.894 34 BB0603 0.017 0.9640.956 0.971 35 OspC_K 0.012 0.877 0.869 0.885 36 BBA36 0.053 0.878 0.8690.888 37 BBM27 0.016 0.874 0.866 0.882 38 BBL40 0.024 0.834 0.821 0.84739 BB0359 0.036 0.872 0.863 0.882 40 BBR42 0.015 0.912 0.904 0.921 41BBJ24 0.012 0.911 0.904 0.919 42 BB0543 0.009 0.883 0.877 0.889 43BB0774 0.011 0.882 0.877 0.888 44 BB0365 0.014 0.935 0.927 0.943 45BB0844 0.061 0.846 0.834 0.859 46 BBN39 −0.010 0.790 0.777 0.804 47BBK12 0.019 0.804 0.788 0.820 48 BBA07 0.047 0.837 0.827 0.847 49 BBK070.018 0.830 0.818 0.842 50 BBA57 0.053 0.851 0.841 0.862 51 BB0323 0.0220.867 0.860 0.875 52 BBB16 0.016 0.861 0.852 0.871 53 BB0681 0.014 0.8300.823 0.836 54 BBA03 0.016 0.849 0.839 0.859 55 BBB09 0.005 0.823 0.8120.835 56 BBA66 0.002 0.881 0.872 0.890 57 BB0238 0.020 0.848 0.844 0.85358 BBA48 0.025 0.825 0.817 0.833 59 BB0408 0.032 0.805 0.798 0.812 60BBK53 0.013 0.827 0.816 0.837 61 BBR35 0.034 0.828 0.815 0.840 62 BBS410.021 0.829 0.818 0.839 63 BB0286 −0.044 0.825 0.815 0.835 64 BB03850.015 0.885 0.876 0.893 65 BBG18 0.022 0.819 0.809 0.828 66 BBA15 0.0100.765 0.756 0.774 67 BBC06 0.021 0.840 0.832 0.848 68 BB0181 0.004 0.8000.791 0.808 69 BB0328 0.034 0.820 0.809 0.832 70 BBP39 0.017 0.797 0.7890.806 71 BBK13 0.018 0.806 0.791 0.821 72 BBK52 0.002 0.802 0.788 0.81673 BB0751 0.018 0.779 0.771 0.787 74 BBA63 0.022 0.779 0.765 0.793 75BBL39 −0.006 0.833 0.825 0.842 76 BBL03 −0.007 0.775 0.765 0.785 77BBO40 −0.002 0.799 0.790 0.807 78 BBQ04 −0.002 0.791 0.774 0.807 79BBA19 0.002 0.751 0.742 0.760 80 BB0668 0.051 0.795 0.785 0.804 81 BBM360.007 0.785 0.774 0.796 82 BBN28 0.050 0.764 0.750 0.778 83 BB0649 0.0190.816 0.810 0.823 84 BBS30 0.004 0.827 0.818 0.835 85 BB0811 0.029 0.7540.738 0.770 86 BB0476 0.031 0.768 0.755 0.780 87 BBK23 0.030 0.764 0.7500.778 88 BB0260 −0.077 0.767 0.756 0.778 89 BB0056 0.022 0.737 0.7230.751 90 BB0048 0.032 0.746 0.735 0.757 91 BB0108 0.014 0.804 0.7920.817 92 BBA40 0.016 0.746 0.738 0.755 93 BB0337 0.029 0.777 0.769 0.78594 BBE09 0.007 0.756 0.741 0.770 95 BB0652 0.013 0.739 0.723 0.756 96BBQ19 0.016 0.717 0.703 0.730 97 BBQ35 0.009 0.797 0.786 0.808 98 BBQ130.026 0.746 0.734 0.758 99 BBC10 0.017 0.699 0.690 0.708 100 BBC03 0.0170.737 0.726 0.748 101 BBR28 0.017 0.756 0.748 0.764 102 BB0357 0.0130.766 0.752 0.780 103 BBO20 0.027 0.736 0.727 0.745 104 BBK40 0.0190.733 0.721 0.745 105 BB0772 0.019 0.697 0.683 0.710 106 BB0628 −0.0030.710 0.700 0.720 107 BBM38 −0.031 0.788 0.775 0.802 108 BBG25 0.0280.724 0.714 0.734 109 BBE10 0.022 0.727 0.715 0.739 110 BBJ34 0.0190.752 0.742 0.762 111 BBM28 0.001 0.733 0.719 0.747 112 BBA61 0.0160.720 0.706 0.733 113 BBA70 0.014 0.729 0.713 0.746 114 BBR12 0.0330.704 0.691 0.718 115 BB0384 0.011 0.733 0.719 0.747 116 BB0805 0.0290.709 0.694 0.725 117 BBA10 0.010 0.729 0.715 0.744 118 BB0502 0.0260.691 0.674 0.708 119 BBK50 0.016 0.727 0.715 0.739 120 BBB12 0.0210.714 0.699 0.730 121 BBA04 0.002 0.688 0.675 0.700 122 BB0144 0.0280.714 0.700 0.729 123 BBN33 0.024 0.732 0.721 0.744 124 BBA58 0.0320.687 0.676 0.698 125 BBO16 0.031 0.690 0.682 0.698 126 BB0159 0.0170.743 0.734 0.752 127 BBP26 0.020 0.692 0.681 0.703 128 BBQ40 0.0320.690 0.674 0.705 129 BBK46 0.007 0.723 0.710 0.736 130 BBE02 0.0360.746 0.732 0.759 131 BBA20 0.002 0.687 0.678 0.695 132 BBQ54 0.0250.710 0.698 0.722 133 BBH32 0.017 0.699 0.683 0.714 134 BB0028 0.0160.712 0.703 0.722 135 BBG23 0.012 0.686 0.674 0.698 136 BB0461 0.0260.674 0.659 0.689 137 BBA68 −0.045 0.670 0.659 0.681 138 BB0651 0.0190.719 0.705 0.733 139 BBD08 0.016 0.652 0.642 0.661 140 BBJ40 0.0150.717 0.702 0.731 141 BBL28 0.001 0.712 0.702 0.722 142 BB0039 −0.0130.697 0.681 0.714 143 BBS36 0.028 0.676 0.659 0.692 144 BBF01 −0.0260.683 0.670 0.695 145 BB0142 −0.002 0.718 0.707 0.730 146 BBN20 0.0360.657 0.645 0.669 147 BB0214 −0.020 0.689 0.680 0.697 148 BBQ76 0.0170.688 0.672 0.704 149 BB0739 0.019 0.671 0.662 0.680 150 BBN12 0.0240.657 0.647 0.668 151 BBS26 −0.001 0.673 0.665 0.681 152 BB0517 0.0050.656 0.646 0.666 153 BBQ08 0.023 0.674 0.663 0.685 154 BBN26 0.0150.661 0.653 0.669 155 BBG01 0.092 0.645 0.627 0.663 156 BBM03 −0.0170.662 0.651 0.673 157 BBF25 0.017 0.654 0.642 0.665 158 BBJ26 0.0200.648 0.634 0.663 159 BBJ25 0.017 0.647 0.640 0.654 160 BB0424 0.0180.673 0.658 0.688 161 BBH29 0.023 0.639 0.628 0.650 162 BBE19 0.0160.664 0.652 0.675 163 BBF13 0.012 0.657 0.644 0.669 164 BBO02 0.0020.645 0.631 0.659 165 BB0150 0.001 0.649 0.637 0.662The top Orfs discriminate very well. The first nine Orfs all have an AUCof >0.95, and further down the rank, the ability diminishes. The 25thimmunogen has an AUC of 0.90, the 50th immunogen has an AUC of 0.85, the100th immunogen has an AUC of 0.74, and the 165th Orf has an AUC of0.65, which still exceeds the upper 95% confidence interval for randomexpectations for the AUC.

To extend the analysis to combinations of antigens, kernel methods andsupport vector machines were used, as described by Vapnik (92), to buildlinear and nonlinear classifiers. Different kernels, including linear,polynomial, and radial basis function, were evaluated. Only the radialbasis function kernel showed an increase in the AUC when noise wasadded, and accordingly, this kernel was chosen for subsequentsimulations in which noise was introduced. For each data set, thesupport vector machines were tuned using a wide parameter sweep toachieve the best gamma and cost values. Results were validated with 10runs of threefold cross-validation. As input to the classifier, thehighest-ranking 2, 5, 25, and 45 Orfs were used on the basis of eitherBayes-regularized ANOVA F scores or frequencies of later LB seraexceeding a 3-SD cutoff. The results of two ranking schemes weresimilar, and only the frequency ranking results are shown in FIG. 7.

For the present data set, there were negligible differences in the ROCcurves obtained using 2, 5, 25, or 45 antigens. The mean AUC values overthe 10 validation runs were >0.98 for two antigens and a perfect 1.0 forfive or more antigens. The unsurpassable performance in this experimentwith relatively few antigens may be attributed to the highdiscrimination provided by the first several antigens on the list bythemselves. In a realistic diagnostic setting with sera coming fromvarious sources and backgrounds and with interoperator variances, onemight expect some addition of noise in the data. To further examine howcombinations of antigens increase the discriminatory power, twodifferent noise models and their effects on the classifiers wereexplored. The noise model involves the addition of uniform Gaussiannoise. Each point (u) in the data set has some noise added such thatu′=u+N(μ=0, σ²=s), where s is constant across the whole data set. Noiselevels are generated by scaling s. In general, using more antigens inthe classifier increases resistance of the simulated assay to noise. Allof the classifiers discriminate very well with low noise levels. For thetwo-antigen classifier, the AUC dropped to the value expected by chanceby the time noise was at a scale of 75. The five-antigen classifiervalue dropped to 0.6 with a noise level of 150. The 25- and 45-antigenclassifiers still performed relatively well, with mean AUC values of0.74 and 0.71, respectively. Hence, based on the criteria of highpredictive value and robustness in the face of increasing noise, 25antigens were as informative as 45 antigens.

DISCUSSION

The genome-wide protein array for B. burgdorferi allowed comparison offar more proteins than could be compared previously with one-dimensionalWestern blots (8, 24, 33). While comparable numbers of proteins foranalysis might theoretically be obtained with two-dimensionalelectrophoresis (66), scarce immunogens in the lysates would beoverlooked. Moreover, unless the microbe's cells were taken directlyfrom an infected animal, informative antigens that were expressed onlyin vivo would be not be included from samples subjected toelectrophoresis. This Example of natural infections of humans andwhite-footed mice with B. burgdorferi followed genome-wide arrayanalyses of antibody responses to poxvirus infections in humansimmunized with a smallpox vaccine and to F. tularensis infections inexperimental animals (29, 35, 87) and ELISA format studies of T.pallidum ORFs (11, 61). The major emphasis of the previous studies wasidentification of immunogens after immunization with whole microbes orduring infection. That same goal was pursued in this Example in thestudy of natural infections that occurred in two very differentecological settings: (i) patients with different stages of Lyme disease,including the arthritis in later disease, and (ii) white-footed mice,which are a major reservoir host of B. burgdorferi in the United Statesand in which infection is nearly universal in enzootic areas. Asdiscussed below, the goal of discovery of new antigens was met: many newimmunogens were identified among the Orfs ofB. burgdorferi.

Of equal interest was a second question: how many of the predictedproteins of this pathogen elicit an antibody response during naturalinfection? For this, the concern was the set of proteins that were notdemonstrably immunogenic. Only by including most of a genome's ORFs inthe experiment could one address this question, which as a generalprinciple is relevant to many other infectious diseases. Important forhypothesis testing for this second goal was the likelihood of falsenegatives or type II errors. If minimizing false positives or type Ierrors (i.e., inaccurately identifying an Orf as an immunogen) was theexperimental design challenge for the first goal, then minimizing falsenegatives (i.e., overlooking Orfs that were truly immunogenic) was thechallenge for the second goal. In the present study, type II errorscould happen for several reasons.

Indisputably, failure to amplify, clone, and then express a given ORFwould lead to a miss of an Orf that was actually immunogenic. Of the˜20% of the Orfs that were absent from the array, undoubtedly someelicit antibody responses during infection. But in many of these cases,the missing Orf was a member of a PF, at least one member of which wasrepresented in the array. Other ORFs were not included because they hadcharacteristics of pseudogenes. Taking these considerations intoaccount, it was estimated that at least 90% of the nonredundant ORFsthat were true genes were included in the array analysis. When calledfor, some missing ORFs were successfully amplified in reattempts usingeither the original primers or modifications of the primers. In theseinstances, addition of the antigens missing from first array to a secondarray did not materially change the results. This suggests that returnsdiminish as further efforts to fully constitute the array consumegreater resources.

Another basis for type II errors would be posttranslationalmodifications that are important for antibody recognition that occur inB. burgdorferi but not in E. coli. While one cannot rule out alimitation to the study for this reason, there is no evidence or onlyscant evidence that glycosylation or a similar posttranslationalmodification affects antigenicity in Borrelia spp. The most prevalentprotein modification in Borrelia spp. appears to be the addition of alipid moiety to the N terminus of the processed proteins in a fashiontypical of many types of bacteria. While E. coli cells are capable ofcarrying out this lipidation function for recombinant Borrelia proteins,this activity did not occur in the acellular transcription-translationreactions used here. This indicates that the significantly greaterrepresentation of lipoproteins among immunogens than that expected basedon a lipoprotein's size among all Orfs was not attributable toantibodies to the lipid moieties themselves. Instead, the comparativelygreater immunogenicity of lipoproteins may be a consequence of themitogenicity and adjuvantlike qualities of bacterial lipopeptides. Forthe 1,292 B31 ORFs that were successfully amplified, cloned, andexpressed, some of the products may have been overlooked as immunogensbecause their epitopes are conformational and proper folding was notachieved in the in vitro reaction or subsequently when the polypeptidewas printed. This possibility cannot be rule out. But the correct callsfor the well-established antigens included in the array, such as OspC,FlaB, P66, P83/100, BmpA, fibronectin-binding protein, and VlsE, amongothers, as “immunogens” indicate that there were few instances of typeII errors on the basis of loss of conformational epitopes or some otherartifact of the procedures.

Another limitation of the study, at least in the case of the human sera,was the restriction of secondary antibodies to antibodies that werespecific for IgG. By failing to account for IgM antibody binding, thetotal number instances in which the Orfs were recognized by antibodiesduring early infection may have been under estimated. However, it is notsuspected that this effect was great if it occurred at all. There was noinstance of an Orf that was recognized by antibodies in sera from earlyinfection and not by antibodies obtained later in the disease. Therationale for limiting antibody detection to IgG was the generallypoorer specificity of IgM-based assays for Lyme disease (34, 91). Theimportance of eventually evaluating antigens for their predictive valuewith IgM as well as IgG antibodies is recognized, but the focus here wason identification of immunogens with the greatest informative value(that is, with high specificity as well as sensitivity). Notwithstandingthe actual and theoretical limitations of the study, we concluded thatthe array results were not confined to identification of new immunogensbut could also be used to gauge the proportion of proteins that are notimmunogens. As far as it is known, this perspective on immune responsesduring natural infection is unique among studies of proteomes ofbacteria, fungi, or parasites. By taking this perspective, it wasestimated that the number of Orfs that elicited antibodies in at leastsome individuals that were infected was about 200, or ˜15% of the 1,292Orfs subjected to analysis with two panels of sera representingdifferent stages of infection. Three types of data supported thisconclusion: the magnitude of sign differences between pairs of LBpatient sera with control sera (see Table 2), the number of Orfs withcorrected, regularized P values <0.01, and number of Orfs with areasunder the ROC curve that exceeded the upper confidence limit for randomexpectations (see Table 5). Of this larger set of immunogens, ˜100 werebroadly enough reactive across several LB serum samples that they couldbe used to distinguish groups of infected individuals from groups ofcontrols. This interpretation also seemed to hold true for white-footedmice, which generally recognized the same subset of proteins as humans.The absolute number of distinct (i.e., non-cross-reactive) antigens isprobably less than the first accounting suggested, because of the heavyrepresentation of proteins in PFs on the immunogen list (Table 1). Theseveral Bdr proteins on the list could probably be replaced in an arrayby one or two Bdr proteins with no loss of sensitivity.

TABLE 2 Pairwise comparisons of reactivities of sera with proteome arrayof B. burgdorferi Controls Early LB Later LB Mean (95% CI) Mean (95% CI)Mean (95% CI) Reference differences in sign differences in signdifferences in sign clinical between serum between serum between serumgroup n pairs^(a) P^(b) pairs^(a) P^(b) pairs^(a) P^(b) Controls 18 −7.1(−32 to 18) 0.5 −188 (−217 to −159) <10⁻⁵ Early LB 24  −7 (−37 to 23)0.9 −198 (−213 to −184) <10⁻⁶ Later LB 19 106 (36 to 177) <10⁻⁵  109 (35to 184) <10⁻⁵ ^(a)The sera were panel 1 sera from controls and frompatients with early and later LB. CI, confidence interval.^(b)Determined by the exact Wilcoxon signed-rank test.

The question of the minimal set of antigens necessary for discriminationbetween sera from patients and sera from controls was also addressed bythe ROC curve analysis (FIG. 7). The introduction of increasing levelsof noise provided a rough simulation of applying an assay in practice(that is, in different locations, at different times, and with differentoperators). It also allowed assessment of the effect of differentamounts of heterogeneity in the total population. By this measure, 25antigens provided more robustness in the face of increasing noise than 2or 5 antigens, while expansion of the set to 45 antigens providedmarginal if any advantage over 25 antigens.

To sum up, it was determined that proteins that detectably elicitantibodies during natural infection constitute about 15% of thepolypeptides that might be expressed. In certain embodiments,incorporation of 2% of the total Orfs in an assay appears to besufficient to provide very high levels of sensitivity and specificity.The attention now turns to what the high-value immunogens are. In thecourse of this study, it was discovered that several protein antigens ofB. burgdorferi that have promise for serodiagnosis of LB but which wereunappreciated as immunogens during infection. These previously unknownantigens appear to be as informative as other proteins, such as FlaB,OspC, P66, BmpA, and VlsE, that have established value for LBserodiagnosis. In addition, in this study we also rediscovered severalother proteins that may have been observed in a limited number ofstudies to be immunogenic in either natural or experimental infectionsbut whose value had not been confirmed or which had not been furtherdeveloped. Among these are the Bdr proteins.

The list of immunogenic proteins identified by proteome array analysiswas compared with lists of genes that were more highly expressed undervarious conditions simulating infection in the natural hosts and werereported by Revel et al. (74), Ojaimi et al. (67), Brooks et al. (13),and Tokarz et al. (88). The concurrence between the proteome list andthe four DNA array lists was greatest for the study of Revel et al., andaccordingly, this study was the study used for comparison. Revel et al.employed three experimental conditions: (i) 23° C. and pH 7.4 in brothmedium, which represented the environment in the unfed tick; (ii) 37° C.and pH 6.8 in broth medium, which represented the environment in ticksas they are feeding on a host and transmitting B. burgdorferi; and (iii)a dialysis chamber in the peritoneum of rats. Of the 79 Orfs that showeda 2-fold increase in mRNA under fed-tick conditions in comparison tounfed-tick conditions, the following 23 (29%) were among thehigh-ranking immunogens: BB0323, BB0329, BB0365, BB0668, BB0681, BB0844,BBA03, BBA07, BBA25, BBA34, BBA36, BBA66, BBB19, BBI42, BBK07, BBK13,BBK32, BBK53, BBL40, BBM27, BBO40, BBP39, and BBQ03. Four of these Orfsare encoded by the lp36 plasmid. Among the 19 Orfs whose expression wasfound by Revel et al. to significantly increase in dialysis chambers incomparison to conditions mimicking unfed ticks, 5 (26%) were on theantigen list. The only three Orfs whose expression decreased underconditions associated with mammalian infection were BBA15 (OspA) andBBA16 (OspB), whose expression was known to decrease in the fed ticksand during early infections in mammals (32, 80, 81), and BB0385 (BmpD).Thus, there was an association between the upregulation of genes in thefed ticks and mammals and the immunogenicity of the gene products ininfected humans.

Western blots of two-dimensional electrophoresis gels provide greaterresolution than one-dimensional gels and allow detection of lessabundant immunogens in lysates. Nowalk et al. performed such anproteomic analysis with the same samples that constituted serum panel 1(66). Fifteen of the 21 proteins identified by Nowalk et al. asimmunogens were also high-ranking Orfs in the present study. Theseproteins include four Erp proteins (BBL39, BBL40, BBN38, and BBP39),three oligopeptide ABC transporters belonging to PF 37 (BB0328, BB0329,and BBB16), two PF 54 proteins (BBA64 and BBA66), a RevA protein(BBM27), and the unique hypothetical protein BBA03, as well as theestablished antigens BB0147 (FlaB), BB0365 (LA7), BB0603 (P66), andBBA15 (OspA).

The large number of proteins newly identified as immunogenic precludesdiscussion of each of them in depth here. Instead, we limit our remarksto the Bdr proteins (PF 113), flagellar apparatus proteins, and BBK07and BBK12, the two members of PF 59. Of all the PF proteins, the Bdrproteins were the most prevalent among the Orfs shown in Table 1. Itwaspreviously reported that LB patients, but not controls, hadantibodies to some of the Bdr proteins (99), but in that study we didnot include BdrT (BBG33), the highest-ranked Bdr protein here. Whileproteins in PFs tend in general to be more immunogenic than other non-PFOrfs, if only because of their multiple versions in a cell, the Bdrproteins may be doubly immunogenic because they have intramolecularrepeats as well (98). The number of copies of the peptide TKIDWVEKNLQKDor a variation of this peptide in a Bdr sequence determines the size ofthe protein. The BBG33 protein, which is 266 amino acids long, is thelargest Bdr protein encoded by the B31 genome. Most of the other Bdrproteins are less than 200 residues long. If the internal repeats areimmunodominant epitopes, then BdrT would display more of these repeatsfor the binding of antibodies than other Bdr proteins and, consequently,generate higher spot intensities. The coefficient for BBL27 regressed onBBG33 is 0.86, and the y intercept is −0.29 (FIG. 3), an indication oflower levels of binding across all sera to the shorter Bdr. BdrT, alsocalled BdrF2, has been reported to be upregulated in “host-adapted” B.burgdorferi and to be specifically expressed during early infection inmice (75, 97).

This study revealed that several flagellar apparatus proteins besidesFlaB flagellin (BB0147), the FlgE hook protein (BB0283), and the FlaAprotein (BB0668) (42, 51, 69, 77) elicit antibody responses duringinfection. Brinkmann et al. found that FlgE of T. pallidum wasfrequently bound by antibodies in sera from patients with syphilis (11).FliL (BB0279) stood out among this larger group of flagellar antigensbecause of the frequency with which it was recognized by both human andwhite-footed mouse serum. Indeed, the field mice had antibody to FliLmore frequently than they had antibody to FlaB, the long-standingflagellar antigen of choice for diagnosis. FliL has 178 residues and isthe flagellar basal body-associated protein, which as an inner membraneprotein interacts with the cytoplasmic ring of the basal body of theflagellum apparatus. Among all organisms, the most similar proteinsoutside the genus Borrelia are the FliL proteins of T. pallidum,Treponema denticola, and Leptospira interrogans, but the sequenceidentities with the proteins of these other spirochete species are lessthan 35%. In comparison, the FlaB protein of B. burgdorferi is 40%identical to the homologous flagellin proteins of Treponema spp. As acomponent of an immunoassay, FliL may show less antigeniccross-reactivity with the homologous proteins of other bacteria than hasbeen the case with FlaB (59, 73). Of all the newly identified Orfs, themost attention was paid to BBK07 and BBK12. As determined by stringentcriteria, these are predicted lipoproteins, and although the amino acidsequences are 88% identical, the ORFs are located several ORFs apart inthe left arm of the lp36 plasmid. Comparison of the BBK07 and BBK12 genesequences of strain B31 with the sequences of two other strains, 297 andN40, revealed >98% sequence identity between the strains for thesesequences, an indication that a single example of each could be used todetect antibodies to other strains of B. burgdorferi. Although BBK12and, by inference, BBK07 are expressed by cells cultivated in thelaboratory (FIG. 6), neither had previously been identified as anantigen. This may be attributable in part to the tendency of the lp36plasmid to be lost sooner than other plasmids from B. burgdorferi duringserial cultivation (7, 72, 79); thus, this plasmid may have frequentlybeen absent from the lysates that investigators used for Westernblotting and other fractionations in pursuit of diagnostic antigens. Butanother reason why BBK12 and BBK07 may have been overlooked is thatthese genes appear to be unique to B. burgdorferi. They have not beenfound to date in the two other major Lyme disease agents: Borreliaafzelii and Borrelia garinii. Using a DNA array comprising variouslipoprotein genes of B. burgdorferi, Liang et al. did not find evidenceof the BBK07 and BBK12 genes in either B. afzelii or B. garinii (55).Glockner et al. reported that they “did not find counterparts of the B.burgdorferi plasmids lp36 and lp38 or their respective gene repertoirein the B. garinii genome” (43). Searches of all deposited GenBanksequences of B. garinii and B. afzelii, including two genomes of eachspecies, likewise did not reveal a PF 59 ortholog. As determined by thisanalysis, the lp34 plasmid of B. afzelii has orthologs of B. burgdorferiORFs in the order BBK01-BBK13-BBK15-BBK17-BBK21-BBK22-BBK23-BBK24, butBBK07 and BBK12 are absent from this and other replicons. If thisgenetic difference between LB species is confirmed, it suggests thatBBK07 or BBK12 can be used in serological assays to distinguish B.burgdorferi infections from B. afzelii and B. garinii infections. Thesegenetic distinctions between lineages may also provide insight intodifferences in pathogenesis and clinical manifestations between LBspecies.

Estimation of the Number of Immunogenic Orfs.

The size of the set of proteins that were immunogenic in B. burgdorferiinfections was assessed by examining the relative amounts of binding forantibodies in panel 1 serum specimens and each of the 1,292 strain B31Orfs. To do this, the sign for each possible pair of sera in the panelwas determiend. The member of a pair that had the higher intensity valuefor a given Orf was assigned a value of “1,” and the pair member withlower reactivity was assigned a value of “0.” As a hypothetical example,if serum a had an intensity value of 3,246 for Orf x and serum b had avalue of 1,711 for Orf x, then serum a was assigned a value of “1” andserum b was assigned a value of “0” for the pairwise comparison in thematrix.

Under the null hypothesis, a given serum sample would have the highervalue of the pair in one-half of the comparisons, or 646 comparisons inthis case. This was observed when controls were compared to controls,early-infection sera were compared to early-infection sera, andlater-infection sera were compared to later-infection sera; the observedmean values were 646 (95% confidence interval, 482 to 810), 646 (95%confidence interval, 524 to 768), and 646 (95% confidence interval, 505to 787), respectively. In contrast to these results for within-grouppairs were the results for between-group pairs, e.g., a control serumand an LB serum. Table 2 summarizes the intergroup comparisons. Excessbinding in the range from 100 to 200 Orfs was also observed withlater-infection sera compared to early-infection sera. From theseresults, it was estimated that the upper limit for immunogenic Orfsduring human infection was 200, or 15% of the 1,292 strain B31 Orfs onthe array.

Simulation to Establish a Cutoff Frequency.

The mean and SD for each Orf with all control serum samples in eachpanel were determined. Then, for each Orf and for every serum sample ineach panel, the number of SDs above or below the mean for the controlsera in the same serum panel for the Orf in question was determined fornormalization. For each sample all the Orfs that had array spots withvalues that were 3 SDs above the mean for the negative controls in theexperiment for the given Orf were tabulated and summed. The frequenciesof each Orf that appeared in this cumulative list were then determinedTo provide an exact test of the significance of the counts that wereobtained, the linkages were randomized for a given normalized value andan Orf and then likewise counted the times that an Orf was associated bychance with an SD that was 3 above the controls. This gave an estimateof the distribution under random conditions. Four replicates wereperformed, and the means were used to provide a distribution under thenull hypothesis of random association between SD and Orf. FIG. 8 showsthe means and confidence intervals for the four replicates, i.e., whatwas expected under random expectations. This is compared to what weobserved with 39 sera from patients with later LB.

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All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inchemistry and molecular biology or related fields are intended to bewithin the scope of the following claims.

We claim:
 1. A method of detecting exposure of a patient to Borreliausing a patient sample comprising: a) contacting a patient sample with aBBK07 and/or BBK12 protein, wherein said BBK07 protein is capable ofbinding a BBK07 patient antibody, and wherein said BBK12 protein iscapable of binding a BBK12 patient antibody, and b) detecting thepresence or absence of said BBK07 and/or BBK12 patient antibody in saidsample based on binding, or lack thereof, of said BBK07 protein to saidBBK07 patient antibody or said BBK12 protein to said BBK12 patientantibody, thereby detecting exposure of said patient to Borrelia.
 2. Themethod of claim 1, wherein said Borrelia is Borrelia burgdorferi.
 3. Themethod of claim 1, wherein said contacting is with said BBK07 protein,and wherein said detecting detects the presence or absence of said BBK07patient antibody in said sample.
 4. The method of claim 1, wherein saidcontacting is with said BBK12 protein, and wherein said detectingdetects the presence or absence of said BBK12 patient antibody in saidsample.
 5. The method of claim 1, wherein said contacting is with bothsaid BBK07 protein and said BBK12 protein, and wherein said detectingdetects the presence or absence of both said BBK07 patient antibody andsaid BBK12 patient antibody in said sample.